Novel targets of acinetobacter baumannii

ABSTRACT

The present invention provides antigenic polypeptides expressed during an infection by a pathogenic organism, such as Acinetobacter and compositions comprising these polypeptides. The invention further provides compositions for use in treating, preventing or detecting a bacterial infection, in particular vaccine compositions using the antigenic polypeptides. The invention further provides antibodies directed to said antigenic polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application from Ser. No. 15/427,976(filed Feb. 8, 2017), which is a Divisional application from Ser. No.14/362,058 (filed May 30, 2014), which was a 371 application ofPCT/EP2012/004939, Novel Targets of Acinetobacter Baumannii, by SimonUrwyler, et al, (filed Nov. 29, 2012), and which claims priority to andbenefit of: European Patent Application 11191320.8 (filed Nov. 30,2011). The full disclosure of the prior application is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to antigenic polypeptides expressed duringan infection by a pathogenic organism, such as Acinetobacter andcompositions comprising these polypeptides. The invention furtherrelates to their use in treating, preventing or detecting a bacterialinfection, in particular the use of the antigenic polypeptides invaccination. The invention further relates to antibodies directed tosaid antigenic polypeptides.

BACKGROUND OF THE INVENTION

Acinetobacter spp. are widely distributed in nature. The genusAcinetobacter is divided into about 20 species. They are gram-negative,oxidase-negative, non-motile, nitrate-negative, non-fermentativebacteria.

Acinetobacter baumannii is the most frequently isolated species in thisgenus. They are able to survive on various surfaces (both moist and dry)in the hospital environment. A. baumannii has only recently beenrecognized as a nosocomial pathogen. Invasive techniques such assurgery, and pulmonary ventilation combined with immunocompromisedpatients, have led to the increased importance of the Acinetobactergenus as nosocomial pathogens.

The frequencies of both nosocomial and community-acquired infectionshave increased steadily over the years. In addition, treatment of theseinfections has become more challenging due to the emergence of(multi)-drug resistant strains.

Acinetobacter infections are usually diagnosed through symptoms foraerobic bacterial infections in combination with microbial cultures ofbody fluids originating from the infected tissue. The cultured bacteriaare then identified in vitro. A variety of genotypic methods has beenexplored and applied to investigate the diversity or phylogeny in thegenus. These methods include high-resolution fingerprinting with AFLP,PCR-RFLP with digestion of PCR amplified sequences, and analysis ofvarious DNA sequences.

One of the most important developments in recent medical history is thedevelopment of vaccines which provide prophylactic protection from awide variety of pathogenic organisms. Many vaccines are produced byinactivated or attenuated pathogens which are injected into anindividual. The immunized individual responds by producing both ahumoral (antibody) and cellular (cytolytic and/or helper and/orregulatory T cells etc) response.

However the use of attenuated organisms in vaccines for certain diseasesis problematic due to the lack of knowledge regarding the pathology ofthe condition and the nature of the attenuation. An alternative to theuse of inactivated or attenuated pathogens is the identification ofpathogen epitopes to which the immune system is particularly sensitive.In this regard many pathogenic toxins produced by pathogenic organismsduring an infection are particularly useful in the development ofvaccines which protect the individual from a particular pathogenicorganism.

A so-called subunit vaccine presents an antigen to the immune systemwithout introducing pathogenic particles, such as viruses, whole orotherwise. Mostly such subunit vaccines are produced by recombinantexpression of an antigen in a host organism, purification from the hostorganism and preparation of a vaccine composition.

In general, Acinetobacter species are considered nonpathogenic tohealthy individuals. The recently recognized clinical importance ofAcinetobacter species has stimulated interest in understanding thevarious bacterial and host components involved in the pathogenesis ofthese diseases. The knowledge of the interaction plays an important rolein controlling the infection. Acinetobacter infections usually involveorgan systems that have a high fluid content (e.g. respiratory tract,CSF (cerebrospinal fluid), peritoneal fluid, urinary tract), manifestingas nosocomial pneumonia, infections associated with continuousambulatory peritoneal dialysis (CAPD), or catheter-associatedbacteriuria.

Pantophlet et al. describe O antigens of Acinetobacterlipopolysaccharides (LPS) and corresponding antibodies foridentification of Acinetobacter isolates (Pantophlet R. et al., Clinicaland Diagnostic Laboratory Immunology, 9, 60-65 (2002)).

Tomarasz et al. identified the polycistronic csuAB gene cluster andshowed its importance in the production and assembly of pili as well asin the subsequent formation of biofilms, e.g. on hospital surfaces andmedical devices (Tomarasz A. P. et al., Microbiology, 154, 3398-3409(2008)).

U.S. Pat. No. 6,562,958 discloses about 4000 nucleic acid and amino acidsequences relating to A. baumannii, however, they are mostly withunidentified function. U.S. Pat. No. 6,713,062 discloses OmpA and OmpAlike protein being capable of stimulating gastrin and IL-8 geneexpression.

However, no vaccines were developed as of today. Vaccines based onsurface-exposed and secreted proteins against Acinetobacter infectionshave not been developed yet due to a lack of availability of feasibletargets.

Therefore, there is a high medical need in the art for antigenicpolypeptides expressed during an infection by Acinetobacter, preferablyA. baumannii, and which are suitable for vaccine development and whichare feasible for production of diagnostic, prophylactic and therapeuticantibodies.

A number of methods have been developed to identify potential antigenicpolypeptides from various pathogens, however, they do not provide ageneral tool to prove the suitability of such polypeptides asimmunogenic target in a vaccine composition.

Accordingly, the technical problem underlying the present invention isto provide clinically prevalent A. baumannii targets to be used in avaccine composition and/or for production of diagnostic, prophylacticand therapeutic valuable antibodies.

The technical problem is solved by the provision of nucleic acidsencoding antigenic polypeptides and antibodies or antibody-bindingfragments that bind the antigenic polypeptides.

SUMMARY OF THE INVENTION

The present invention provides a vaccine composition comprising at leastone polypeptide encoded by a nucleic acid molecule comprising apolynucleotide selected from the group consisting of:

-   -   a) a polynucleotide having the nucleic acid sequence depicted in        any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 15;    -   b) a polynucleotide encoding a fragment, analog or functional        derivative of a polypeptide encoded by the polynucleotide of        (a), wherein said fragment, analog or functional derivative has        immunostimulatory activity;    -   c) a polynucleotide encoding a polypeptide having an amino acid        sequence that is at least 80% identical to the amino acid        sequence depicted in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12,        14, and 16 and having immunostimulatory activity;    -   d) a polynucleotide which is at least 80% identical to the        polynucleotide of (a), and which encodes a polypeptide having        immunostimulatory activity;    -   e) a polynucleotide which hybridizes under stringent conditions        to the polynucleotide of any one of (a) to (d); and    -   f) a polynucleotide that is complementary to the full length of        a polynucleotide of any of (a) to (d).

Preferably said nucleic acid molecule is genomic DNA.

In one embodiment of the invention, said polypeptide is derived from thegenus Acinetobacter; preferably said polypeptide is derived from thespecies Acinetobacter baumanii.

In another embodiment of the invention, the vaccine composition furthercomprises a pharmaceutically acceptable carrier and/or adjuvant.

In another embodiment, the present invention provides an antigenicpolypeptide consisting of an amino acid sequence depicted in any one ofSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16; or fragment, analog orfunctional derivative thereof, wherein said fragment, analog orfunctional derivative has immunostimulatory activity.

In further embodiments, the present invention provides a nucleic acidmolecule encoding the antigenic polypeptide of the invention, anexpression vector comprising said nucleic acid molecule and a host cellcomprising said vector and/or said nucleic acid of the invention.

In a further embodiment, the present invention provides an antibody oran antigen-binding fragment thereof that specifically binds theantigenic polypeptide of the invention, wherein said antibody orantigen-binding fragment thereof is capable of inducing an effectorfunction towards Acinetobacter baumanii. The antibody provided by theinvention is polyclonal or monoclonal; preferably human. Said antibodymay be N-terminally, internally and/or C-terminally modified, such as byoligomerization, and conjugation to a drug and/or a label.

The monoclonal antibody or an antigen-binding fragment thereof of theinvention preferably is capable of inducing an effector function towardsAcinetobacter baumanii. Most preferably, the monoclonal antibody of theinvention or an antigen-binding fragment thereof specifically binds theepitope consensus motif PVDFTVAI shown in SEQ ID NO: 36.

The monoclonal antibody of the invention is preferably produced from ahuman B cell or a hybridoma obtained by fusion of said human B cell witha myeloma or heteromyeloma cell. The invention thus provides a hybridomacapable of producing the monoclonal antibody of the invention. Theinvention further provides a nucleic acid encoding the light chain andthe heavy chain of the inventive antibody and a vector comprising saidnucleic acid as well as a host cell comprising said vector and/or saidnucleic acid.

In a further embodiment, the present invention provides a method forproducing the monoclonal antibody of the invention comprising culturingthe hybridoma as defined herein under conditions allowing for secretionof an antibody, and optionally purifying the antibody from the culturesupernatant.

In a further embodiment, the present invention provides a pharmaceuticalcomposition comprising the antigenic polypeptide or the antibody of theinvention and a pharmaceutically acceptable carrier. In a furtherembodiment, the present invention provides a diagnostic compositioncomprising the antigenic polypeptide or the antibody of the inventionfor detecting a bacterial infection in a patient. The antibody of theinvention is provided for use in the treatment, prevention and/ordetection of a bacterial infection in a mammal; preferably a human.

In a further embodiment, the present invention provides a polypeptidefor use in the treatment and/or prevention of a bacterial infection in amammal encoded by a nucleic acid molecule comprising a polynucleotideselected from the group consisting of:

a) a polynucleotide having the nucleic acid sequence depicted in any oneof SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15;

b) a polynucleotide encoding a fragment, analog or functional derivativeof a polypeptide encoded by the polynucleotide of (a), wherein saidfragment, analog or functional derivative has immunostimulatoryactivity;

c) a polynucleotide encoding a polypeptide having an amino acid sequencethat is at least 80% identical to the amino acid sequence depicted inany one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16 and havingimmunostimulatory activity;

d) a polynucleotide which is at least 80% identical to thepolynucleotide of (a), and which encodes a polypeptide havingimmunostimulatory activity;

e) a polynucleotide which hybridizes under stringent conditions to thepolynucleotide of any one of (a) to (d); and

f) a polynucleotide that is the complement of the full length of apolynucleotide of any of (a) to (d).

Preferably the mammal is human. In a further embodiment of the presentinvention the bacterial infection to be treated, prevented and/ordetected is caused by Acinetobacter baumanii, said bacterial infectionmay be hospital-acquired. The antigenic polypeptide compositions for useaccording to invention may further comprise a delivery vehicle;preferably a virosome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows IgG titres in sera from convalescent A. baumannii patients(left) and ordinary, randomly selected blood donors (right).

Antigenic polypeptides according to the invention were recombinantlyexpressed, purified and tested by ELISA with sera from convalescent A.baumannii patients and ordinary, randomly selected blood donors indifferent dilutions. Numbers within the charts reflect the number ofsera tested and reacting with the antigenic polypeptide (A-H) at adilution as indicated by the different colours given in the legend.

Titres are defined as the highest serum dilution that generates anantigen specific ELISA signal twice the signal of the correspondingblank. The majority of patient sera tested contain antibodies againstthe targets identified by the present invention. The patient seracontain generally higher titers compared to healthy blood donors. Forall antigens, individual patient sera could be identified with extremelyhigh antibody titers (≥ 1/6400), proving that the antigens areimmunogenic in human and are expressed during infection. This stronglyindicates that these newly identified targets are feasible for vaccinedevelopment and generation of prophylactic/therapeutic antibodies.

A: His-AB023 corresponding to SEQ ID NO: 2; B: His-AB024 correspondingto SEQ ID NO: 4; C: His-AB025 corresponding to SEQ ID NO: 6; D:His-AB030 corresponding to SEQ ID NO: 8; E: His-AB031L1 corresponding toSEQ ID NO: 10; F: His-FimA corresponding to SEQ ID NO: 12; G: His-CsuABcorresponding to SEQ ID NO: 14; H: His-OmpA corresponding to SEQ ID NO:16.

FIG. 2 shows an ELISA from rabbit sera.

Rabbits were immunized with a recombinant his-tagged antigenicpolypeptide. Final bleed and pre-immune sera were tested via ELISA onELISA plates coated individually with the different antigenicpolypeptides. In addition the final bleeds were tested via ELISA onplates coated with control reagents: His-tagged OmpA which served as acontrol for A-G and His-CsuAB which served as a control for H. TheFigures show that the major immune response is caused by the target andnot by the His-tag which is present on the control as well. Comparableresults were obtained with a duplicate set of immunized rabbits. Theimmune and preimmune sera dilutions used were:

A: α-His-AB023 (1:6400); B: α-His-AB024 (1:6400); C: α-His-AB025(1:6400); D: α-His-AB030 (1:25600); E: α-His-AB031L1 (1:12800); F:α-His-FimA (1:400); G: α-His-CsuAB (1:3200); H: α-His-OmpA (1:6400).

FIG. 3 shows an immunoblot analysis.

The specificity of the rabbit antisera was tested. Cell lysates fromvarious A. baumannii (AB) and P. aeruginosa (PA) strains as negativecontrols, respectively were prepared, proteins separated on SDS-PAGE andblotted onto nitrocellulose. Rabbit sera against the differentpolypeptides (immune sera) and pre-immune sera were used at a dilution1:1000 (experimental details given in Example 6).

Bacterial lysates: 1: AB: ATCC19606 wild type; 2: AB: ATCC19606 OmpAK.O; 3: AB: ATCC19606; CsuE K.O; 4: PA O11; 5: AB: AB-N; 6: AB: Luh8168;7: AB: Ruh134; 8: AB: SAN;

Immune-sera: A: α-His-AB023; B: α-His-AB024; C: α-His-AB025; D:α-His-CsuAB; E: α-His-OmpA; F: α-His-AB030; G: α-His-FimA; H:α-His-AB031L1;

FIG. 4 shows another immunoblot analysis.

The specificity of the rabbit antiserum specific for the polypeptideFimA was tested within culture supernatant. FIG. 4 shows arepresentative immunoblot of an A. baumannii (AB-Non-mucoid), a P.aeruginosa (PA O11) and an E. coli (DH5a) strain.

Overnight bacteria cultures were centrifuged and the proteins within thesupernatant precipitated. The cell pellets (P) and precipitatedsupernatant (SN) of equivalent culture volumes were examined byimmunoblot analysis for the presence of FimA using □-His-FimA rabbitantiserum. A total of 29 A. baumannii strains were analyzed byimmunoblotting for the presence of FimA within the supernatant as wellas the bacterial pellet. 45% contained detectable amounts in the cellpellet while 55% contained detectable amounts in the SN.

AB: A. baumannii strain AB-NM (Non-mucoid); PA: P. aeruginosa O11; EC:E. coli DH5α.

FIG. 5 shows another immunoblot analysis.

The specificity of the selected human sera was tested by immunoblotanalysis. Recombinant proteins were separated on SDS-PAGE and blottedonto nitrocellulose. Different patient sera (A-F) were used against thedifferent polypeptides (1-7) at a dilution of 1:500 (experimentaldetails are given in Example 6). To exclude artefacts of antibodiesdirected against the His-tag, combinations of recombinant antigens werechosen to include with each immunoblot a His-tagged protein as negativecontrol that is not recognized by the corresponding patient serum.

Recombinant proteins: 1—His-AB023; 2—His-AB024; 3—His-AB025;4—His-AB030; 5—His-FimA; 6—His-CsuAB; 7—His-OmpA; 8—AB031-L1 (no humansera identified yet for AB031 L1 on immunoblots).

FIG. 6 shows a FACS analysis; wherein

picture A shows FACS analysis of A. baumannii strains ATCC19606 wildtype (wt), OmpA KO (OmpA-) and CsuE KO (CsuE-) using a patient sera at adilution of 1:200. Bacterial population was gated using forward andsideward scatter and 20,000 bacteria were measured;

picture B shows FACS analysis of A. baumannii strains ATCC19606 wildtype (wt) using the same patient sera and instrument settings as in A.Patient serum was used without (S) or with recombinant OmpA (S+rOmpA) asinhibitory agent; and

picture C shows an immunoblot analysis using patient sera of celllysates of A. baumannii ATCC19606 wild type (1), OmpA KO (2) as negativecontrol and CsuE KO (3). Ponceau stain of blot confirms equal loading ofcell lysates. The protein band of OmpA in cell lysates of ATCC19606 wildtype and CsuE KO is apparent as well with the Ponceau stain.

FIG. 7 relates to another FACS analysis; wherein

picture A shows FACS analysis of A. baumannii, strains ATCC19606 (wt)and CsuE-KO (CsuE-) with indirectly fluorescence labelled □CsuAB rabbitimmune serum (IS) or corresponding preimmune serum (PIS). As secondaryantibody, FITC labelled goat-anti-rabbit-IgG was used. Histogram chartsblotting the fluorescence signal intensity to number of events wasprepared from gated bacteria. Bacterial population was gated usingforward and sideward scatter and 5,000 bacteria were measured.

picture B shows FACS analysis of different A. baumannii strains (ATCC19606, CsuE KO, Luh9415, Ruh134, Ruh875). The chart shows the percentageof bacteria that were indirectly fluorescence labelled with □CsuABrabbit immune serum (IS) or corresponding preimmune serum (PIS).Bacteria were considered positive with a FL1-H signal intensity of >20.

FIG. 8 shows the results of an agglutination assay and animmunofluorescence analysis; wherein

picture A shows an agglutination of live A. baumannii (Strain ATCC19606)using 1.5 mg/ml total rabbit IgG, purified from □□CsuAB rabbitimmuneserum or naive rabbit serum; and

picture B shows an immunofluorescence analysis of A. baumannii (StrainsATCC 19606 and CsuE KO). Bacteria were grown on glass slides for 24 h incell culture medium (IMDM) containing 10% FCS. Bacteria were labelledwith DAPI to localize bacterial DNA (top Figures) and indirectlyfluorescence labelled using □CsuAB rabbit immune serum (IS) orcorresponding preimmune serum (PIS) with FITC labelled secondaryantibody (bottom Figures).

FIG. 9 shows a bactericidal assay and an immunoblot analysis; wherein

pictures A and B show the bactericidal assay. The charts shows thenumber of colony forming units (cfu) after incubation with purified IgGfrom rabbit CsuAB immune serum (grey bars) or from naive rabbit serum(black bars); wherein

A relates to logarithmic growing A. baumannii, ATCC 19606 and CsuE KO(CsuE-), which were incubated with antibody (0.5 μg/well) for 20 minutesat 37° C. As complement source baby rabbit serum (BRS) was added andincubated for 2 h. Eventually cfu were quantified by plating onto LBA;and

B relates to logarithmic growing A. baumannii Ruh 134, which wasincubated with antibody (5 μg/well) for 20 minutes at 37° C. Ascomplement source baby rabbit serum (BRS) or as control heat inactivatedBRS (HBRS) were added and supplemented with or without HL-60 cells(+HL60) previously transformed to neutrophils. Mixtures were incubatedfurther for 2 h. Eventually cfu were quantified by plating onto LBA.

A and B: error bars show Standard deviation of three independent wells;Student's T-test (equal variance, 2-tailed) show statisticalsignificance of <0.05 for:

ATCC19606/α CsuAB compared with CsuE−/α CsuAB; ATCC19606/α CsuABcompared with ATCC19606/Naive IgG; Ruh134+BRS+HL60/α CsuAB compared withRuh134+HBRS+HL60/α CsuAB; Ruh134+BRS+HL60/α CsuAB compared withRuh134+BRS+HL60/Naive IgG; Ruh134+BRS/α CsuAB compared withRuh134+HBRS/α CsuAB; Ruh134+BRS/α CsuAB compared with Ruh134+BRS/NaiveIgG.

Picture C shows an immunoblot analysis of wild type and CsuE KO A.baumannii of the strain ATCC19606; and

FIG. 10 shows the result of an FimA pulldown assay; wherein total IgG(10 □g) from FimA rabbit immune serum (1) was coated onto Protein Abeads (20 ul bed volume) and used to capture native FimA from A.baumannii culture supernatant (0.4 ml) of the strain Luh9415, known tosecrete FimA into the SN. Equal amounts of total IgG from a naive rabbitserum (2) was used as negative control. Total captured proteins werereleased into SDS-PAGE sample buffer by boiling for 10 min and 7% wereseparated by SDS-PAGE. Native FimA was visualized by immunoblot analysisusing a FimA immuneserum at a dilution of 1:1000.

FIG. 11 shows passive immunization with CsuAB rabbit immune sera.

Neutropenic mice were infected with A. baumannii after i.p. injectionwith either 0.15 ml immune serum (solid lines) or an equal volume ofserum from a naive animal (dashed lines). Survival of mice was recordedfor 4-5 days. The virulence of the A. baumannii strain varied betweendifferent strains and dates of executions. Experiments B and C wereperformed in parallel while the experiment shown in A was performed on aseparate date.

Picture A shows Strain AB-M, 10 animals per group; picture B showsStrain AB-M, 14 animal per group, and picture C shows strain AYE, 14-15animals per group.

FIG. 12 shows an active immunization experiment. Mortality in a modelfor A. baumannii induced pneumonia model after active immunization. Micewere vaccinated with antigens (solid lines A-F: A: AB025—9 animals, B:AB030—10 animals, C: AB031L1—9 animals, D: FimA—9 animals, E: CsuAB—10animals, F: OmpA—9 animals) and pneumonia was induced afterwards byintra-tracheal inoculation of A. baumannii (strain AB-M). As a control,a group of mice was vaccinated with the adjuvant only (dashed line A-G;10 animals). In a second control group PBS was used instead of a vaccineor adjuvant (solid line G; 9 animals). For all antigens tested (A-F), abeneficial effect of the vaccine compared to the adjuvant control groupwas observed. A statistically significant effect was observed for AB030,while the other antigens just missed the threshold of 5% for statisticalsignificance. Two reasons might contribute to this effect. Firstly, thelow number of animals and, secondly the lower mortality of the controlgroups (G), as compared to previous experiments. The mortality was mostlikely lower because the animals in active immunization experiments aremuch older than those used in passive immunization. This is due to theduration of the active immunization protocol of several weeks.

FIG. 13 shows a passive immunization experiment. Mice were renderedtransiently neutropenic by intra-peritoneal injection ofcyclophosphamide on days 4 and 3 before A. baumannii inoculation. On day0, 3 h before A. baumannii inoculation, mice were passively vaccinatedintraperitoneally with either 0.15 ml rabbit antiserum, naïve rabbitserum or PBS. Pneumonia was induced analogous to the active immunizationprotocol. Survival, clinical score and body weight were monitored.

DETAILED DESCRIPTION

According to the present invention a vaccine composition is providedcomprising at least one polypeptide encoded by a nucleic acid moleculecomprising a polynucleotide selected from the group consisting of

a) a polynucleotide having the nucleic acid sequence depicted in any oneof SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 15;

b) a polynucleotide encoding a fragment, analog or functional derivativeof a polypeptide encoded by the polynucleotide of (a), wherein saidfragment, analog or functional derivative has immunostimulatoryactivity;

c) a polynucleotide encoding a polypeptide having an amino acid sequencethat is at least 80% identical to the amino acid sequence depicted inany one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16 and havingimmunostimulatory activity;

d) a polynucleotide which is at least 80% identical to thepolynucleotide of (a), and which encodes a polypeptide havingimmunostimulatory activity;

e) a polynucleotide which hybridizes under stringent conditions to thepolynucleotide of any one of (a) to (d); and

f) a polynucleotide that is complementary to the full length of apolynucleotide of any of (a) to (d).

The polypeptides of the invention, as referred to herein, are summarizedin Table 1 below:

TABLE 1 Polypeptide Amino acid sequence Nucleic acid sequence AB023 SEQID NO: 2 SEQ ID NO: 1 AB024 SEQ ID NO: 4 SEQ ID NO: 3 AB025 SEQ ID NO: 6SEQ ID NO: 5 AB030 SEQ ID NO: 8 SEQ ID NO: 7 AB031 SEQ ID NO: 10 SEQ IDNO: 9 FimA SEQ ID NO: 12 SEQ ID NO: 11 CsuAB SEQ ID NO: 14 SEQ ID NO: 13OmpA SEQ ID NO: 16 SEQ ID NO: 15

The term “fragment” as used herein refers to any fragment of thepolypeptide as defined herein which has immunostimulatory activity. Thefragment has a minimum length of at least 4, 8, 15, 20, 30, 50, 100amino acids. It is preferred that the fragment comprises an epitope of6-8 amino acids in length, a minimal length of 4-5 amino acids and amaximal length of 15 amino acids to the total length of the proteindepicted in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16.

An “analog of a polypeptide” is meant to refer to a moleculesubstantially similar in function to either the entire molecule or to afragment thereof.

The term “functional derivative” of a polypeptide means a polypeptidewith a similar structure and the same biological function.

The term “immunostimulatory activity” as used herein, refers to inducingan initial immune response to an antigen. Preferably, the polypeptidehaving immunostimulatory activity as defined herein is capable ofinducing an immune response against infection with Acinetobacter, mostpreferred the polypeptide of the invention is capable of inducing animmune response against infection with A. baumannii. The term ‘immuneresponse’ as used herein refers to a change in antibody content in anybody fluids, which are reactive with the polypeptides, as well aschanges in cellular responses to the polypeptides, such as T-cells andcells of the innate immune system, as well as changes in inflammatorymarkers like cytokines and chemokines and other immunological markersindicative of a modulation of normal immune functions. The immuneresponse against these pathogenic organisms was monitored with ELISA,immunoblot and the like.

The “sequence identities” as referred herein of related polypeptides andpolynucleotides can be determined by means of known procedures. Asequence identity of the related polypeptides to the antigenicpolypeptides depicted in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,16 of at least 75%, more preferably 80% or 85%, and most preferred 90%or 95% is envisaged. As a rule, computer programs with algorithms takingaccount of the special requirements are used. For the purposes of thepresent invention, the computer program used for the determination ofthe identity between two sequences is BLASTP (for comparison of aminoacid sequences) and BLASTN (for comparison of nucleotide sequences), asdescribed e.g. by Altschul S et al., Nucl Acid Res 25: 3389-3402 (1997).The BLAST programs can be obtained from the National Centre forBiotechnology Information (NCBI) and from other sources (e.g. BLASTHandbook, Altschul S et al., NCB NLM NIH Bethesda Md. 20894; Altschul Set al., J. Mol. 215: 403-410 (1990)). For the purposes of the presentinvention, the BLASTN and BLASTP algorithm with the following defaultsettings is used:

BLASTN: Scoring Parameters:

Match/Mismatch Scores 1, −3; Gap costs: Existence: 5, Extension: 2;Filters and Masking: Low complexity regions selected; Mask for lookupTable only selected; Mask lower case letters not selected

BLASTP: Scoring Parameters:

Matrix: BLOSUM62; Gap costs: Existence: 11, Extension: 1; Compositionaladjustments: Composition-based statistics 2; Filters and Masking: Noneselected; Program Advanced Options; −G Cost to open gap [Integer];default=5 for nucleotides 11 proteins; −E Cost to extend gap [Integer];default=2 nucleotides 1 proteins;−q Penalty for nucleotide mismatch[Integer]; default=−3;−r reward for nucleotide match [Integer];default=1; −e expect value [Real]; default=10; −W wordsize [Integer];default=11 nucleotides 3 proteins; −y Dropoff (X) for BLAST extensionsin bits (default if zero); default=20 for BLASTN 7 for other programs,−X X dropoff value for gapped alignment (in bits); default=15 for allprograms except for BLASTN for which it does not apply; −Z final Xdropoff value for gapped alignment (in bits); 50 for BALSTN 25 for otherprograms.

For sequence comparison, the complete polypeptide sequence (SEQ ID NO: 2or 4, 6, 8, 10, 12, 14 and 16, respectively) is used as the sequence towhich a related sequence is compared. Specifically, to determine theidentity of a polypeptide with unknown homology to e.g. the polypeptidewith SEQ ID NO: 2 according to the invention, the amino acid sequence ofsaid first polypeptide is compared to the amino acid sequence of thepolypeptide shown in SEQ ID NO: 2, over the entire length of SEQ ID NO:2. Similarly, to determine the identity of a polynucleotide with unknownhomology to e.g. polynucleotide with SEQ ID NO: 1 according to theinvention, the nucleic acid sequence of said first polynucleotide iscompared to the nucleic acid sequence shown in SEQ ID NO: 1, over theentire length of SEQ ID NO: 1.

Standard “stringent conditions” for hybridization are disclosed inAusubel et al. (Eds.), Current Protocols in Molecular Biology, JohnWiley & Sons (2000). Exemplary stringent hybridization conditionsinclude washes with 0.1×SSC/0.1% SDS for 15 min at 68° C.

The present invention provides a vaccine composition as defined above,wherein the nucleic acid molecule encoding a polypeptide is genomic DNA.

The nucleic acid sequences encoding the polypeptides of the presentinvention can be amplified by PCR from genomic DNA of an A. baumanniistrain using primers containing appropriate restriction sites forcloning.

According to the present invention the vaccine composition comprises atleast one polypeptide wherein said polypeptide is derived from the genusAcinetobacter.

More preferably the vaccine composition comprises at least onepolypeptide wherein said polypeptide is derived from the speciesAcinetobacter baumanii.

The terms “Acinetobacter baumannii” or “A. baumannii” as used hereinrefer to Acinetobacter baumanii species as classified in AcinetobacterMolecular Biology, 2008, Ed.: Ulrike Gerischer, Caister Academic Press.Examples are A. baumannii strains SDF, AYE, ATCC 19606, ACICU Ruh134,Ruh875, AB-M, AB-NM and SAN, whose references and sources are describedin Table 6. . References and information regarding taxonomy and strainscan be received on the Pubmed homepage.

A. baumannii causes different types of infections including, amongothers, pneumonia, bacteremia, and skin and soft tissue infections. Overthe last decades A. baumannii has emerged as a pathogen of increasingclinical importance due to the global increase in the incidence ofinfections caused by this organism. Infections caused by this pathogenhave been especially problematic in patients receiving mechanicalventilation and burn patients. A. baumannii can cause outbreaks inintensive care units and trauma/burn units, which are presumably causedby passage of the organism from infected or colonized individuals andcontaminated hospital equipment to uninfected patients.

The results shown in Table 2, below prove that the targets identified bythe present invention are representative of all A. baumannii clinicalisolates tested so far. Strain SDF represents the only A. baumanniistrain which is not a clinical isolate but was isolated from body lice.This strain is lacking the genes for FimA and CsuAB.

Table 2 below shows the percentage of amino acid identity of proteinsencoded by different A. baumannii strains. Amino acid sequences encodedby the A. baumannii genome AB307, corresponding to the polypeptidesidentified by the present invention (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,16), were compared to 13 other sequenced genomes. In case of the antigenAB031 only the extracellular loop L1 was used for comparison.

TABLE 2 Conservation of amino acid identity by various A. baumanniistrains ATCC ATCC Target AB307 AB056 AB057 AB058 AB059 SDF AYE 1797819606 ACICU AB900 6013113 6013150 6014059 AB023 100% 100% 100% 100% 100%99% 100% 99% 99% 100% 99% 100%  99% 100% AB024 100% 100% 100% 100% 100%86% 100% 99% 99%  99% 99% 100% 100%  99% AB025 100% 100% 100% 100% 100%90% 100% 93% 91%  91% 88% 100% 100%  91% AB030 100% 100% 100% 100% 100%99% 100% 99% 99%  99% 99% 100% 100%  99% AB031 100% 100% 100% 100% 100%100%  100% 100%  100%  100% 97% 100% 100% 100% L1* FimA 100% 100% 100%100% 100% — 100% 74% 100%  100% 94% 100% 100% 100% CsuAB 100% 100% 100%100% 100% — 100% 100%  100%  100% 100%  100% 100% 100% OmpA 100% 100%100% 100% 100% 89% 100% 93% 94%  93% 93%  99%  99%  93% *loop comparedonly. — no homologues detected

The high degree of amino acid identity of the proteins within various A.baumannii strains shows the broad specificity of the antigenic proteinsand confirms their high therapeutic value. The high prevalence of thegenes indicates that the protein is important, possibly essential,during the life cycle of the bacteria. Therefore the protein is likelyexpressed during infection. The high degree of conservation pointsincreases the chance to induce an immune response or to identify apolyclonal or monoclonal antibody capable of binding most or possiblyall clinically relevant A. baumannii strains. Additionally, the highdegree of amino acid conservation indicates that mutations of thesegenes are rare, thus reducing chances for rescue mutants duringtherapeutic treatment.

The present invention provides a vaccine composition as defined hereinwherein said vaccine composition further comprises a pharmaceuticallyacceptable carrier and/or adjuvant.

The term “adjuvant” as used herein refers to a substance distinct fromtarget antigen that is capable of increasing the antigenic response. Theadjuvant may be selected from Freund's adjuvants (complete andincomplete), Gerbu adjuvant (GERBU Biotechnik GmbH, Germany),mycobacteria such as BCG, M. vaccae, or Corynebacterium parvum, Choleratoxin or tetanus toxoid, E. coli heat-labile toxin, quil-saponinmixtures such as QS-21 (SmithKline Beecham), MF59 (Chiron) and variousoil/water emulsions (e.g. IDEC-AF), MALP-2, ISCOMs. Other adjuvantswhich may be used include, but are not limited to: mineral salts ormineral gels such as aluminium hydroxide, aluminium phosphate, andcalcium phosphate; surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, keyhole limpet hemocyanins, anddinitrophenol, immunostimulatory molecules, such as saponins, muramyldipeptides and tripeptide derivatives, short nucleic acid stretches suchas CpG dinucleotides, CpG oligonucleotides, monophosphoryl Lipid A, andpolyphosphazenes, particulate and microparticulate adjuvants, such asemulsions, liposomes, virosomes, virus-like particles, cochleates, orimmunostimulating complex adjuvants. Cytokines are also useful due totheir lymphocyte stimulatory properties. Many cytokines useful for suchpurposes will be known to one of ordinary skill in the art, includinginterleukin-2 (IL-2), IL-12, GM-CSF and many others. Furthermore ligandsfrom the chemokine family, such as RANTES, a lipoprotein, a lipopeptide,a yeast cell wall component, a double-stranded RNA, a bacterialcell-surface lipopolysaccharide (LPS), flagellin, a U-richsingle-stranded viral RNA, a suppressor of cytokine signalling smallinterfering RNA (SOCS siRNA), a Pan DR epitope (PADRE) and mixturesthereof are suitable.

The definition of “pharmaceutically acceptable carrier” is meant toencompass any carrier, which does not interfere with effectiveness ofthe biological activity of the active ingredient and that is not toxicto the host to which it is administered.

Accordingly, one or more polypeptides of the invention or fragments,analogs and functional derivatives thereof may be used to prepare aprophylactic or therapeutic vaccine for administration to an individualin need thereof. Such a vaccine which contains one or more polypeptidesof the present invention, as the principal or member active ingredient,can be administered in a wide variety of therapeutic/prophylactic dosageforms in the conventional vehicles for topical, mucosal (nasal, oral),systemic, local, and parenteral administration. Thus, the inventionprovides compositions for parenteral administration which comprise asolution of a polypeptide according to the invention optionally incombination with a suitable adjuvant and/or equivalent delivery vehiclesdissolved or suspended in an acceptable carrier, preferably an aqueouscarrier. A variety of aqueous carriers may be used, e.g., water,buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.These compositions may be sterilized by conventional, well knownsterilization techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, among manyothers. Actual methods for preparing parenterally administrablecompounds will be known or apparent to those skilled in the art and aredescribed in more detail in for example, Remington: The Science andPractice of Pharmacy (“Remington's Pharmaceutical Sciences”) Gennaro A Red. 20th edition, 2000: Williams & Wilkins PA, USA.

The route and regimen of administration will vary depending upon thestage or severity of the condition to be treated, and is to bedetermined by the skilled practitioner. For example, the polypeptide(s)according to the invention and compositions containing it can be usedfor preparing a pharmaceutical composition that can be administered insubcutaneous, intradermal, or topical or mucosal or intramuscular form.All of these forms are well known to those of ordinary skill in thepharmaceutical arts.

Advantageously, suitable formulations of the present invention may e.g.be administered in a single dose, which may be repeated daily, weekly,or monthly.

Initial doses can be followed by booster doses, following immunizationprotocols standard in the art. The immunostimulatory effect of thecompositions and methods of the instant invention can be furtherincreased by combining any of the above-mentioned polypeptides,including their combination with delivery vehicles and/or with an immuneresponse potentiating compound Immune response potentiating compoundsare classified as either adjuvants or cytokines. Adjuvants may enhancethe immunological response by providing a reservoir of antigen(extracellularly or within macrophages), activating macrophages andstimulating specific sets of lymphocytes.

Each of the inventive polypeptides can be conjugated to a proteinous ornon-proteinous delivery vehicle. Examples of such conjugations aredescribed in Szaóo R. et al., (Biochim Biophys Acta. 2010 December;1798(12):2209-16. Epub 2010 Jul. 24.) and in “Conjugation of haptens”(Lemus & Karol, Methods Mol Med. 138:167-82, 2008). It is preferred thatthe delivery vehicle itself has an immune effect, which means thedelivery vehicle itself is immunogenic.

The delivery vehicle is selected from the group consisting ofimmunogenic peptides, immune stimulation nucleic acid sequences like GPCislands, limpet hemocyanin (KLH), tetanus toxoid (TT), cholera toxinsubunit B (CTB), bacteria or bacterial ghosts, liposome, chitosome,virosomes, microspheres, dendritic cells, virus-like particles or theirlike.

In another embodiment, the present invention provides a vaccinecomposition further comprising a delivery vehicle as defined hereinabove. Preferably, the delivery vehicle is a virosome.

The antigenic polypeptides, compositions, or formulation thereofaccording to the present invention may be delivered via the deliveryvehicles defined herein above, preferably by a virosome.

The prophylactic or therapeutic compositions of the present inventionare for administration in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents. The preparations ofthe invention are administered in effective amounts. An effective amountis that amount of a pharmaceutical preparation that alone, or togetherwith further doses, stimulates the desired response. Generally, doses ofimmunogens ranging from 0.01 μg/kilogram to 500 μg/kilogram body weight,depending upon the mode of administration, are considered effective. Thepreferred range is believed to be between 0.1 μg/kilogram and 10μg/kilogram body weight. The absolute amount will depend upon a varietyof factors, including the composition selected for administration,whether the administration is in single or multiple doses, andindividual patient parameters including age, physical condition, size,weight, and the stage of the disease. These factors are well known tothose of ordinary skill in the art and can be addressed with no morethan routine experimentation.

The dosage regimen utilizing the compositions of the present inventionis selected in accordance with a variety of factors, including forexample species, age, weight, and medical condition of the patient, thestage and severity of the condition to be treated, and the particularcompound thereof employed. A physician of ordinary skill can readilydetermine and prescribe the effective amount of the vaccine required toprevent, counter, or arrest the progress of an infectious disease.Optimal precision in achieving a concentration of a drug with the rangethat yields efficacy either without toxicity or with acceptable toxicityrequires a regimen based on the kinetics of the drug's availability totarget sites. This process involves a consideration of the distribution,equilibrium, and elimination of the drug, and is within the ability ofthe skilled practitioner.

In the uses of the present invention, the compounds herein described indetail can form the active ingredient and are typically administered inadmixture with suitable pharmaceutical diluents or excipients suitablyselected with respect to the intended form of administration, that is,oral tablets, capsules, elixirs, syrups, and the like, and consistentwith conventional pharmaceutical practices. For instance, foradministration in the form of a tablet or capsule, the active vaccinecomponent can be combined with a non-toxic pharmaceutically acceptableinert carrier such as ethanol, glycerol, water and the like. Moreover,when desired or necessary, suitable binders, lubricants, disintegratingagents and coloring agents can also be incorporated into the mixture.Suitable binders include, without limitation, starch, gelatin, naturalsugars such as glucose or beta-lactose, corn sweeteners, natural andsynthetic gums such as acacia, tragacanth or sodium alginate,carboxymethyl cellulose, polyethylene glycol, waxes and the like.Lubricants used in these dosage forms include, without limitation,sodium oleate, sodium stearate, magnesium stearate, sodium benzoate,sodium acetate, sodium chloride and the like. Disintegrators include,without limitation, starch, methyl cellulose, aga, bentonite, xanthangum and the like.

For parenteral administration, sterile suspensions and solutions aredesired. Isotonic preparations which generally contain suitablepreservatives are employed when intravenous administration is desired.Intraesophageal preparations containing the active drug component can beadmixed with a variety of carrier materials well known in the art, suchas, for example, alcohols, aloe vera gel, allatoin, glycerine, vitaminsA or E oils, mineral oil, PPG2 myristyl propionate, and the like, toform, for example, alcoholic solutions, topical cleansers, cleansingcreams, gels, foams, and lotions, in cream or gel formulationsespecially suited for mucosal applications.

The antigenic polypeptides, compositions, or formulation thereof of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, andcross-linked or amphipathic block copolymers of hydrogels.

In the case the polypeptide according to the invention is used forpreparing a pharmaceutical composition for treating an infectiousdisease, such as an infection caused by A. baumannii, the desiredresponse is control of the infection and/or clearance of the antigenicpolypeptide from the system. In the case of prophylaxis, the desiredresponse is protective immunity to such polypeptide, as measured byimmune responses upon exposure to the antigenic polypeptide. Thesedesired responses can be monitored by diagnostic methods such as ELISA,immunoblot and the like [Raem A M. Immunoassay. 2007. P. Rauch [ed.]Spektrum Akademischer Verlag, Elsevier Gmbh].

The present invention provides an antigenic polypeptide consisting of anamino acid sequence depicted in any one of SEQ ID NOs: 2, 4, 6, 8, 10,12, 14 and 16; or fragment, analog or functional derivative thereof,wherein said fragment, analog or functional derivative hasimmunostimulatory activity. Said antigenic polypeptides, fragments,analogs and functional derivatives thereof are defined in more detailherein above.

The antigenic polypeptide consisting of an amino acid sequence depictedin any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12 14 and 16 that maycomprises up to 50, 45, 40, 35, 30, 25, 20, 15, 10, preferably up to 5,more preferably up to 3 additional amino acids; or fragment, analog orfunctional derivative thereof, wherein said antigenic polypeptide,fragment, analog or functional derivative thereof has immunostimulatoryactivity.

Amino acids and amino acid residues described herein may be referred toaccording to the accepted one or three letter code referenced in textbooks well known to those of skill in the art, such as Stryer,Biochemistry, 4th Ed., Freeman and Co., New York, 1995 and Creighton,Proteins, 2nd Ed. Freeman and Co., New York, 1993.

As used herein, the terms “peptide” and “polypeptide” are usedsynonymously and in their broadest sense refer to a molecule of two ormore amino acid residues, or amino acid analogs. The amino acid residuesmay be linked by peptide bonds, or alternatively by other bonds, e.g.ester, ether, etc. As used herein, the term “amino acid” or “amino acidresidue” refers to natural and/or unnatural or synthetic amino acids,including both the D or L enantiomeric forms, and amino acid analogs.

Bacterial surface proteins play a fundamental role in the interactionbetween the bacterial cell and its environment. They are involved inadhesion to and invasion of host cells, in sensing the chemical andphysical conditions of the external milieu and sending appropriatesignals to the cytoplasmic compartment, in mounting defenses againsthost responses and in toxicity. Hence, surface proteins are potentialtargets of drugs aimed at preventing bacterial infections and diseases.Moreover, because surface proteins are likely to interact with the hostimmune system, they may become components of effective vaccines.Vaccines based on surface-exposed and secreted proteins are alreadycommercially available for various infectious diseases; however avaccine against Acinetobacter infections has not been developed yet dueto a lack of availability of feasible targets.

Despite the biological relevance of bacterial surface proteins, theircharacterization is still incomplete. This is mostly owing todifficulties in defining the protein composition and topology on thebacterial surface.

To identify new vaccine candidates and targets for antibodies, threedifferent methods were used. Each one selected for particularrequirements for a vaccine and antibody target candidate.

The first method—“Shedome analysis”—uses proteolytic enzymes to “shed”the bacterial surface. The peptides generated are separated from thewhole cells, identified by mass spectrometry and subsequently assignedto proteins using public available databases (Rodriguez-Ortega M J etal., Nature Biotechnology, 24, 191-197, 2006).

To discriminate between contaminants, such as intracellular proteins ofhighly abundant proteins like ribosomal proteins, and putative membranetargets, the identified proteins were analyzed for their localizationwithin the bacteria using public available online tools. See, forexample K. Imai et al., Bioinformation 2(9), 417-421 (2008). Proteinsthat were assigned as extracellular or outer membrane protein wereselected for further analysis. In addition, proteins that were annotatedby the UniprotKB Database as a homologue to known extracellular or outermembrane proteins were selected as well.

The concept of the second method—“Comparative proteomics”—is to focus ontargets whose expression is experimentally confirmed in variousAcinetobacter strains. Proteomics, the study of the proteome, haslargely been practiced through the separation of proteins by twodimensional gel electrophoresis. In the first dimension, the proteinsare separated by isoelectric focusing, which resolves proteins on thebasis of charge. In the second dimension, proteins are separated bymolecular weight using SDS-PAGE. The gel is dyed with CoomassieBrilliant Blue or silver to visualize the proteins. Spots on the gel areproteins that have migrated to specific locations.

The mass spectrometer has augmented proteomics. Peptide massfingerprinting identifies a protein by cleaving it into short peptidesand then deduces the protein's identity by matching the observed peptidemasses against a sequence database.

According to the invention the whole proteome of protein preparationenriched for outer membrane proteins was determined by mass spectrometryof five different A. baumannii strains. The five A. baumannii strainsATCC19606, BMBF65, SDF, ACICU, AYE were selected due to their differentsources of isolation. ATCC19606 is an old A. baumannii isolate from 1948(Hugh R., Reese R., Int. J. Syst. Bacteriol. 17: 245-254, 1967), used bymany research laboratories as a reference strain. AYE is an A. baumanniistrain that was epidemic in France during 2001 (Vallenet et al., PLoSOne 3:E1805-E1805 (2008)). ACICU was isolated during an outbreak inRome, Italy 2005 (Iacono M., et al., Antimicrob. Agents Chemother.52:2616-2625 (2008)).

BMBF-65 was isolated from a patient in Singapore in 2004. SDF is theonly non-clinical isolate of A. baumannii that was isolated from bodylice collected in 1997 in Marseille, France (Vallenet et al., PLoS One3:E1805-E1805 (2008)).

To enrich for putative targets that are present on the extracellularsurface, protein preparations were enriched for outer membrane proteinsprior to MS analysis according to their hydrophilic and hydrophobicproperties. The peptides identified by mass spectrometry were assignedto proteins using publicly available databases and selected according toIT-predictions and literature searches.

The third approach refers to identification of targets that arerecognized by antibodies present in sera of convalescent A. baumanniipatients. Accordingly, protein preparations enriched for outer membrane(OM) proteins, were separated by 2-dimensional gel electrophoresis(2DE). The 2DE constituted of an isoelectric focusing (IEF) followed bySDS-polyacrylamide gel electrophoresis (PAGE) step to resolve the OMproteins. Proteins recognized by patient sera were determined byimmunoblot analysis. To increase chances to identify proteins that areexpressed by various different strains, immunoblots of at least two A.baumannii strains were compared and proteins present in all strainsanalyzed were selected for protein identification by MS-analysis. Theproteins were individually characterized and selected according toIT-predictions and literature searches. Proteins that were identified asA. baumannii protein and predicted to be or annotated as an outermembrane protein were chosen as putative targets. In case prior artpredicted homologues of such targets to be down-regulated or absent inantibiotic resistant A. baumannii strains, these targets were excludedfrom further analysis.

According to an aspect of the invention there is provided at least onepolypeptide identified by the approaches according to the invention.

In a preferred embodiment of the invention, said polypeptide isassociated with infective pathogenicity of an organism, preferably of A.baumannii, according to any previous aspect or embodiment of theinvention.

More preferably said polypeptide is at least one of the amino acidsequences SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16 or fragment, analogor functional derivative thereof.

The targets that were selected for vaccine development fulfill at leasttwo of the following three requirements:

1. The targets are accessible to large molecules (approach 1: surfaceproteins identified by Shedome analysis).

2. The targets are expressed by many A. baumannii strains preferably bystrains which represent important clinical isolates (approach 2:Comparative Proteomics).

3. The targets induce an immune response and are expressed in patientsduring infection (approach 3: specific target identification).

The numbers of potential targets meeting the requirements of eachselection step are specified in Table 3. The potential targets selectedby this method are designated in the final row.

Table 3 below shows the selection process for target identification bydifferent approaches. Each approach focuses on a particular requirementdescribed above. Bold numbers indicate number of proteins that meet therequirements of the corresponding selection step. The details of theselection process are given in Example 1.2.

TABLE 3 Comparative Specific Target Shedome Analyis ProteomicsIdentification Total numbers of >3500 potential targets annotated genesin A. baumannii genomes Experimental selection Proteome determinationProteome determination Comparison of 2DE of tryptic digest of of outermembrane prepara- Immunoblots of outer live A. baumannii tions of 5different membrane preparations 163 A. baumannii strains of 2 different1552 A. baumannii strains using patient sera. 7 1^(st) in silicoselection N/A 363 N/A Proteins identified by 5 strains 2^(nd) in silicoselection 7  30 5 IT prediction: Extracellular proteins Outer membraneproteins with surface located epitopes. 3^(rd) in silico selection 3  64 If available, data from literature IT prediction: high prevalence ofgenes high amino acid sequence conservation Selected targets FimA,CsuAB, AB023, AB024, AB023, AB024, OmpA AB025, AB030, AB025, OmpA AB031,OmpA

IT-Prediction was performed as follows: Protein homology detection andstructure prediction by HMM-HMM-comparison was performed using onlinesoftware tool HHpred, Söding J., p. 951-960, (2005) using the HMMdatabase pdb70_3 Sep11, HHblits as MSA generation method with maximal 3iterations and local Alignment mode.

Table 4 shows the structural homologues of SEQ ID NOs: 2, 4, 6, 8, 10,12, 14 and 16 as determined by databank analysis.

TABLE 4 Antigen SEQ ID Protein Query Template Probability³ Template NO:ID¹ HMM² HMM² [%] E-value⁴ P-value⁵ Description¹ AB023 2wjr 346-417 29-95  93.0 0.25 9.9E−06 NanC - Porin SEQ ID 2o4v 52-417 32-411  92.46.6 0.00026 (E. coli) NO: 2 OprP - Porin (P. aeruginosa) AB024 2zfg97-435 7-340 99.7 4E−12 1.6E−16 OmpF - Porin SEQ ID 2fgq 100-435  3-33299.5 9E−11 3.5E−15 (E. coli) NO: 4 Omp32 - Porin (D. acidovorans) AB0252o4v 116-439  60-411  96.3 0.79 3.1E−05 OprP - Porin SEQ ID 2qtk144-474  88-389  90.4 14 0.00054 (P. aeruginosa) NO: 6 Opdk - Porin (P.aeruginosa) AB030 2qdz 269-906  10-554  100.0 1.4E−45 0 FahC - Omp85 SEQID 3efc 241-543  79-375  100.0 2.5E−31 9.9E−36 (P. pertussis) NO: 8YaeT- Omp85 (E. coli) AB031 1ek9 42-485 2-409 100.0 4.2E−45 0 TolC -channel SEQ ID 1yc9 42-486 34-440  100.0 1.7E−44 0 (E. coli) NO: 10Vcec - channel (V. cholerae) FimA 2jmr 21-177 2-155 100.0 4.2E−301.6E−34 FimF - type I SEQ ID 2jty 16-177 1-159 99.9 1.1E−27 4.3E−32 Pili(E. coli) NO: 12 FimA - type I Pili (UP-E. coli) CsuAB 3me0 40-180 8-12798.3 1.4E−05 5.6E−10 PapD - type I SEQ ID 1ze3 38-180 1-121 97.8 0.000431.7E−08 Pili (E. coli) NO: 14 FimD - type I Pili (E. coli) OmpA 3nb3 1-345 1-344 100.0 0 0 OmpA - (E. coli) SEQ ID 2kgw 208-335  1-128 100.01.6E−28 6.2E−33 Ompatb - NO: 16 (M. tuberculosis) ¹Protein ID ofstructural homologue (http://www.ncbi.nlm.nih.gov/ Wang Y, et al.,Nucleic Acids Res. 2007 January; 35(Database issue): D298-300.)including a short description (Name, function, Species) in the lastcolumn. ²HMM: Hidden Markov Model. Amino acid sequences producinghomology between query and template. The number indicate the positionsof amino acid sequence in the query (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,16) or template (Protein ID) sequence that produces homology.³Probability: “Probability of template to be a true positive.” ⁴E-value:“Expect-value. E-value and P-value are calculated without taking thesecondary structure into account. The E-value gives the average numberof false positives (‘wrong hits’) with a score better than the one forthe template when scanning the database. It is a measure of reliability:E-values near 0 signify a very reliable hit, an E-value of 10 meansabout 10 wrong hits are expected to be found in the database with ascore at least this good.” ⁵P-Value: “The P-value is the E-value dividedby the number of sequences in the database. It is the probability thatin a pairwise comparison a wrong hit will score at least this good.”

Any of the polypeptides with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 hasimmunostimulatory activity.

Table 5 refers to expression of the antigenic polypeptides of theinvention in clinical isolates of A. baumannii. A total of 36 clinicalstrains isolated from blood, urine, cerebrospinal fluid, pus andtracheal aspirates of patients admitted to the hospital—were included inthe study. Such clinical isolates (Example 1.1.4) were used to isolatebacterial lysates or precipitated culture supernatant which after gelelectrophoresis were tested by immunoblot analysis. For the detection ofeach antigenic polypeptide the corresponding rabbit antiserum was used(Example 5).

Table 5 shows the percentages and the actual number of clinical isolatesof A. baumannii wherein any of the individual antigenic polypeptidesidentified (target) was shown to be present or absent by immunoblotanalysis in preparations from bacterial cell pellets.

TABLE 5 Target detected in bacterial cell Number of pellet of clinicalisolates Target clinical isolates present absent AB023 20 100% (20) 0%(0) AB024 20 100% (20) 0% (0) AB025 21 100% (21) 0% (0) AB030 21 100%(21) 0% (0) AB031 24 100% (24) 0% (0) FimA 36  44% (16) 56% (20) CsuAB*36  81% (29) 19% (7)  OmpA 32 100% (32) 0% (0) *Expression levels ofcsuAB varied between different strains. 19% showed no, 24% weak and 62%medium to strong expression.

FIG. 2 shows the polypeptides of the invention having immunostimulatoryactivity. Rabbits were immunized with the polypeptides. The sera ofthese rabbits proved positive for polypeptide specific antibodies.

According to a further aspect of the invention there is provided anucleic acid molecule encoding said antigenic polypeptide(s).

In a further aspect, the present invention relates to a vectorcomprising the nucleic acid molecule according to the invention.Moreover, the present invention relates to a host cell comprising saidvector.

There is a significant amount of published literature with respect toexpression vector construction and production and purification ofrecombinantly expressed polypeptides (Sambrook et al (1989) MolecularCloning: A Laboratory Manual, Cold Spring Harbour Laboratory, ColdSpring Harbour, NY and references therein; DNA Cloning: F M Ausubel etal, Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Further, the present invention provides host cells comprising the vectorand/or the nucleic acid suitable for the expression of the vector. Inthe art, numerous prokaryotic and eukaryotic expression systems areknown wherein eukaryotic host cells such as yeast cells, insect cells,plant cells and mammalian cells, such as HEK293-cells, PerC6-cells,CHO-cells, COS-cells or HELA-cells and derivatives thereof arepreferred. Particularly preferred are human production cell lines. It ispreferred that the transfected host cells secrete the produced antibodyinto the culture medium. If intracellular expression is achieved, thenrenaturation is performed in accordance with standard procedures such asdescribed by Benetti P. H. et al., Protein Expr. Purif. 13(3):283-290(1998).

Production of the polypeptides according to any previous aspect orembodiment of the invention comprise: (i) providing a celltransformed/transfected with a vector according to the invention; (ii)growing said cell in conditions conducive to the manufacture of saidpolypeptides; and (iii) purifying said polypeptide from said cell, orits growth environment.

In a preferred embodiment of the invention said cell is a prokaryoticcell.

Alternatively said cell is a eukaryotic cell selected from: fungal,yeast, insect, algae, mammalian, plant.

The present invention provides an antibody or an antigen-bindingfragment thereof that specifically binds the polypeptide as definedabove, wherein said antibody or antigen-binding fragment thereof iscapable of neutralizing Acinetobacter baumanii.

The term “antigen-binding fragment” means any fragment of the antibodycapable of binding to any of the polypeptides defined by the claims. Thefragment has a length of at least 10, preferably 20, more preferably 50amino acids. It is preferred that the fragment comprises the bindingregion of the antibody. It is preferred that the fragment is a Fab orF(ab′)2 fragment or a mixture thereof.

An antibody mediated “effector function” can be the inhibition of aspecific function of the target antigen, such as the neutralization ofan effect of a secreted bacterial toxin, thereby preventing thedetrimental effects of the toxin on protein interactions, enzymaticfunction, cellular functions, cell integrity, tissue structures andother biological process. Another antibody mediated effector functioncan be the inactivation of the function of a specific bacterial protein,such as a porin and other proteins or structures on the cell surface,thereby affecting the normal bacterial life cycle. Another antibodymediated effector function can consist of activation of immunologicalprocesses, such as activation of complement cascade, induction ofcytokine and chemokine production, activation of cellular components ofthe immune system and other immunological reactions leading to thedestruction and removal of bacterial cells.

In a preferred embodiment of the invention said antibody is a polyclonalor a monoclonal antibody, wherein said antibodies are specific to saidpolypeptide.

In order to produce polyclonal antibodies in a host, such as a mouse orrabbit, the host is immunized with the antigenic polypeptide or fragmentor analog or functional derivative thereof, optionally with an adjuvant.Antibodies to the antigenic polypeptide are subsequently collected fromthe sera of the host. The polyclonal antibody can be affinity purifiedagainst the antigen rendering it specific. Such polyclonal antibodypreparations can also be derived from human donors, either vaccinated,convalescent or normal healthy donors, by plasma fractionating togenerate polyclonal immunoglobulin fractions and further enrichedagainst the antigen rendering it specific.

Such polyclonal antibodies were raised by immunizing rabbits with theantigenic polypeptides AB023, AB024, AB025, AB030, AB031L, ABFimA,ABCsuAB and ABOmpA. Four to eight weeks after immunization blood sampleswere collected and sera tested for presence of polypeptide specificantibodies; see FIGS. 3 and 4.

Polyclonal antibodies recognize many different epitopes. In contrastmonoclonal antibodies are specific for a single epitope. Further detailsregarding antibody structure and their various functions can be foundin, “Using Antibodies: A laboratory manual”, Cold Spring HarbourLaboratory Press, 1999.

In a further preferred embodiment, the antibody of the invention is amonoclonal antibody or an antigen-binding fragment thereof which iscapable of inducing an effector function towards Acinetobacter baumanii.Most preferably, the monoclonal antibody of the invention or anantigen-binding fragment thereof specifically binds the epitopeconsensus motif PVDFTVAI shown in SEQ ID NO: 36.

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin. Incertain embodiments, epitope determinants include chemically activesurface groupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree-dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. Monoclonal antibodies usually bind to these consensusmotifs, which are mostly 5 amino acids in lengths, or 6, 7 or 8 aminoacids in length. In a preferred embodiment the antibody provided by theinvention is monoclonal and specifically binds to an epitope consensusmotif of 8 amino acids in length. In certain embodiments, an antibody issaid to specifically bind an antigen when it preferentially recognizesits target antigen in a complex mixture of proteins and/ormacromolecules. In preferred embodiments, an antibody is said tospecifically bind an antigen when the dissociation constant is less thanor equal to about 10 nM, more preferably when the dissociation constantis less than or equal to about 100 pM, and most preferably when thedissociation constant is less than or equal to about 10 pM.

In a further embodiment the antibody of the invention is human. The term“human” as used herein encompasses any partially or fully human antibodyindependent of the source from which the antibody is obtained. Theproduction of a human monoclonal antibody by a hybridoma is preferred.For example, the human monoclonal antibody consisting of human aminoacid sequence can be obtained from a hybridoma wherein the B-cell is ahuman B-cell. The monoclonal antibody may also be obtained by geneticengineering.

“Humanized” antibodies are also contemplated, as are chimeric antibodiesfrom mouse, rat, or other species bearing human constant and/or variableregion domains, bispecific antibodies, recombinant and engineeredantibodies and fragments thereof. “Humanizing” techniques typicallyinvolve the use of recombinant DNA technology to manipulate DNAsequences encoding the polypeptide chains of the antibody molecule.Early methods for humanizing monoclonal antibodies (MAbs) involvedproduction of chimeric antibodies in which an antigen binding sitecomprising the complete variable domains of one antibody is linked toconstant domains derived from another antibody. Methods for carrying outsuch chimerization procedures are described in EP0120694 (CelltechLimited), EP0125023 (Genentech Inc. and City of Hope), EP-A-0 171496(Rev. Dev. Corp. Japan), EP-A-0 173 494 (Stanford University), and WO86/01 533 (Celltech Limited). Generally these applications discloseprocesses for preparing an antibody molecule having the variable domainsfrom a mouse MAb and the constant domains from a human immunoglobulin.Alternative approaches are described in EP-A 023 9400 (Winter), in whichthe complementary determining regions (CDRs) of a mouse MAb have beengrafted onto the framework regions of the variable domains of a humanimmunoglobulin by site directed mutagenesis using long oligonucleotides.See U.S. Pat. No. 7,262,050 for an example of such methods.

Humanized antibodies can also be obtained from transgenic animals. Forexample, transgenic, mutant mice that are capable of producing a fullrepertoire of human antibodies, in response to immunization, have beendescribed (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA,90:2551 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993).Specifically, the homozygous deletion of the antibody heavy chainjoining region (J(H)) gene in these chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production, andthe successful transfer of the human germ-line antibody gene array intosuch germ-line mutant mice results in the production of human antibodiesupon antigen challenge.

The human amino acid sequence of the human monoclonal antibody preventsthe occurrence of undesired adverse effects such as rejection reactionsor anaphylactic shock.

According to a further preferred embodiment, the antibody according tothe present invention is N-terminally, internally and/or C-terminallymodified. The modification is selected from at least one of the di-,oligo-, or polymerization of the monomeric form e.g. by cross-linkingusing dicyclohexylcarbodiimide. The thus produced di-, oligo-, orpolymers can be separated from each other by gel filtration. Furthermodifications include side chain modifications, e.g. modifications ofε-amino-lysine residues, or amino and carboxy-terminal modifications,respectively. Further modifications include post-translationalmodifications, e.g. glycosylation and/or partial or completedeglycosylation of the protein, and disulfide bond formation. Theantibody may also be conjugated to a label, such as an enzymatic,fluorescent or radioactive label.

The antibody according to the present invention is produced from a humanB cell or a hybridoma obtained by fusion of said human B cell with amyeloma or heteromyeloma cell.

The present invention further provides a hybridoma capable of producinga monoclonal antibody. The production of monoclonal antibodies usinghybridoma cells is well-known in the art. The methods used to producemonoclonal antibodies are disclosed by Kohler and Milstein in Nature256, 495-497 (1975) and also by Donillard and Hoffman, “Basic Factsabout Hybridomas” in Compendium of Immunology V. II ed. by Schwartz,1981.

Alternatively to the hybridoma technology the human monoclonal antibodymay also be obtained by recombinant expression of nucleic acids encodingthe light and heavy chain of the antibody.

Accordingly, the present invention provides a nucleic acid encoding thelight and the heavy chain of the antibody, a vector comprising suchantibodies and a host cell comprising such vector and/or such nucleicacids.

Preferably, a vector according to the invention is selected fromadenoviruses, vaccinia viruses, baculoviruses, SV 40 viruses,retroviruses, plant viruses or bacteriophages such as lambda derivativesor M13 comprises at least one nucleic acid encoding the light chain andat least one nucleic acid encoding the heavy chain. A host celltransformed with said vector and cultured under conditions suitable forrecombinant expression of the encoded antibody chain is capable ofassembling the human monoclonal antibody such that a 3-dimensionalstructure is generated which is equivalent to the 3-dimensionalstructure of a human monoclonal antibody produced by a human B-cell. Ifthe light chain is produced separately from the heavy chain, then bothchains may be purified and subsequently be assembled to produce a humanmonoclonal antibody having essentially the 3-dimensional structure of ahuman monoclonal antibody as produced by a human B-cell.

In addition, a method is provided for producing the antibody as definedabove comprising culturing a hybridoma under conditions allowing forsecretion of an antibody, and optionally purifying the antibody from theculture supernatant.

In addition, pharmaceutical compositions comprising the antigenicpolypeptide as defined above or the antibody as defined above areprovided.

The pharmaceutical composition may further comprise pharmaceuticallyacceptable ingredients known in the art.

Preferably, the pharmaceutical compositions are applied for thetreatment of diseases caused by A. baumannii in infections such asblood-stream infection, pneumonia, chronic bronchitis, local infectionsincluding wound infections and invasive infections of joints, mainly inimmunocompromised patients and/or in patients with compromisedrespiratory function. The pharmaceutical compositions are furtherintended for but not limited to the prophylaxis and/or treatment ofhospital-acquired (nosocomial) infections. Since the main victims of A.baumannii infections are intubated patients, burn victims, patients insurgical and/or medical intensive care units, cancer and AIDS patients,immunocompromised patients, immunosuppressed patients, diabeticpatients, military personal, combat personal and associated supportpersonal, as well as intravenous drug abusers, the pharmaceuticalcompositions are in particular intended for prophylaxis and/or treatmentof diseases caused by A. baumannii in said group of patients.

The pharmaceutical composition may further comprise antibiotic drugs.

The pharmaceutical compositions comprise the antigenic polypeptide orthe antibody in a concentration range of 0.1-30 mg/kg body weight.

The pharmaceutical compositions may be administered in any known mannersuch as intravenous, intra-muscular, intra-dermal, subcutaneous,intra-peritoneal, topical, intra-nasal administration, or as inhalationspray.

A further aspect of the invention refers to a diagnostic compositioncomprising the antigenic polypeptide or the antibody as defined abovefor detecting a bacterial infection in a patient. Detection of abacterial infection, in particular a bacterial infection caused by A.baumannii according to the invention, may be performed on isolatedbacterial DNA, or directly from clinical samples like sputum,broncho-alveolar lavage or tracheal aspiration, usually after dilutionin ultrapure H₂O. Preferred are samples directly obtained from a lunglavage of a human such as a human patient with a pulmonary disorder.Clinical samples might also include bodily materials such as blood,blood sera, urine, tissues and the like. Typically the samples may betaken from wound, burn, lung, and urinary tract infections of humans ormammals. Antigenic polypeptides of the invention may be used to checkfor antibodies in blood sera. Antibodies are suitable for detection ofthe antigenic polypeptide (targets) e.g. in a clinical sample. The highvalue as a diagnostic tool of the antigenic polypeptides or the antibodyspecific thereto is demonstrated in Table 1 and FIG. 3.

The present invention provides a polyclonal or monoclonal antibody foruse in the treatment and/or prevention of a bacterial infection in amammal.

Preferably the mammal is human. The antibody is preferably used fortreatment and or prevention wherein the bacterial infection is caused byA. baumannii, most preferably this infection is hospital acquired.

Disease areas that currently are especially amenable to antibody-basedtreatments include cancer, immune dysregulation, and infection.Depending upon the disease and the biology of the target, antibodiesused for treatment—therapeutic antibodies—can have different mechanismsof action. A therapeutic monoclonal antibody may bind and neutralize thenormal function of a target. For example, a monoclonal antibody thatblocks the activity of the protein needed for the survival of a cancercell causes the cell's death. Another therapeutic monoclonal antibodymay bind and activate the normal function of a target. For example, amonoclonal antibody can bind to a protein on a cell and trigger anapoptosis signal. Finally, if a monoclonal antibody binds to a targetexpressed only on diseased tissue, conjugation of a toxic payload(effective agent), such as a chemotherapeutic or radioactive agent, tothe monoclonal antibody can create a guided missile for specificdelivery of the toxic payload to the diseased tissue, reducing harm tohealthy tissue.

Prophylactic antibodies are guarding from or preventing the spread oroccurrence of disease or infection.

An antibody defined by its structure/sequence has potentiallyprophylactic and therapeutic function depending on the time ofadministration.

Further, the present invention provides a polypeptide for use in thetreatment and/or prevention of a bacterial infection in a mammal encodedby a nucleic acid molecule comprising a polynucleotide selected from thegroup consisting of:

a) a polynucleotide having the nucleic acid sequence depicted in any oneof SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 15;

b) a polynucleotide encoding a fragment, analog or functional derivativeof a polypeptide encoded by the polynucleotide of (a), wherein saidfragment, analog or functional derivative has immunostimulatoryactivity;

c) a polynucleotide encoding a polypeptide having an amino acid sequencethat is at least 80% identical to the amino acid sequence depicted inany one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16 and havingimmunostimulatory activity;

d) a polynucleotide which is at least 80% identical to thepolynucleotide of (a), and which encodes a polypeptide havingimmunostimulatory activity;

e) a polynucleotide which hybridizes under stringent conditions to thepolynucleotide of any one of (a) to (d); and

f) a polynucleotide that is the complement of the full length of apolynucleotide of any of (a) to (d).

Preferably the polypeptide is for use in a mammal. More preferably thepolypeptide is for use in treatment and/or prevention of a bacterialinfection wherein the infection is caused by Acinetobacter baumanii;most preferably the antigenic polypeptide of the invention is for use intreatment and/or prevention of a bacterial infection, wherein theinfection is hospital-acquired.

The invention is further illustrated by reference to specificembodiments described in the Examples and Figures presented below.

EXAMPLES Example 1: Identification of Targets (Antigenic Polypeptides)1.1 Materials

Unless not otherwise specified, chemical defined reagents wereanalytical grade and derived from qualified suppliers, mainlySigma-Aldrich (Buchs, Switzerland).

1.1.1 Bacterial Media

Luria-Bertani broth (LB) consisted of 1% (w/v) tryptone(Fluka/Sigma-Aldrich, Switzerland), 0.5% (w/v) yeast extract (Fluka,),1% (w/v) NaCl Immediately after preparation the LB was autoclaved (121°C. for 20 minutes) and kept sterile at room temperature for up to 3months. For LB-agar (LBA) plates, 0.75% (w/v) agar (Fluka), was added toLB before the media was autoclaved. Afterwards the hot LBA wasdistributed into plastic petri dishes (Sterilin, Cambridge, UK) beforethe media cooled below 50° C. Once the LBA within the petri dishessolidified the LBA plates were kept at 4° C. for up to 3 months.BHI-Agar plates were ordered at Becton Dickinson (Heidelberg, Germany).

1.1.2 Bacterial Strains

Several bacterial strains were used. The most relevant bacterial strainsused to generate the data and the experimental procedures are listed inTable 5. In addition, several clinical isolates of A. baumannii, werereceived from Prof. Seifert (Institute of Medical Microbiology andHygiene, University of Cologne, Germany), Prof. Dijkshoorn (LeidenUniversity Medical Centre, Leiden, NL), Prof. Nordmann, (CentreHospitalier Universitaire der Bicêtre, Service deBactériologie-Virologie, Le Kremlin-Bicêtre cedex, France).

TABLE 6 Strain Species Reference Source ATCC19606 A. baumannii Hugh R.,Reese R. Prof. Luis Actis, Miami Int. J. Syst. Bacteriol. University,Department of 17: 245-254, 1967 Microbiology, 40 Pearson Hall Oxford,Ohio 45056 OmpA KO A. baumannii Gaddy, J. A. et al., Infection andImmunity 77 (8), S. 3150-3160. (2009) CsuE KO A. baumannii Tomares,Microbiology, 154, 3398 (2008) AYE A. baumannii Vallenet et al., Profs.D. Raoult, M. PLoS One 3: E1805- Drancourt URMITE- E1805(2008)) CNRSUMR6236, Marseille France SDF A. baumannii ACICU A. baumannii Iacono M.,et Prof. Alessandra Carattoli al., Antimicrob. Agents Department forInfectious, Chemother. 52: 2616- Parasitic and Immune- 2625(2008).Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299,00161 Rome-Italy Ruh134 A. baumannii Clinical isolate Prof. L.Dijkshoorn, Leiden Rotterdam, NL, 1982 University Medical Centre,Leiden, NL Ruh875 A. baumannii Clinical isolate Dordrecht, NL, 1984Berlin-95 A. baumannii Clinical isolate Berlin, Prof. Seifert, InstituteGE, 2006 of Medical Microbiology and Hygiene, University of Cologne,Germany BMBF65 A. baumannii Clinical isolate Singapore, 2004 AB-M A.baumannii Eveillard, et al., Prof. Marie-Laure Joly Journal of InfectionGuillou, UFR Sciences 60 (2), 154-161, 2010 pharmaceutiques etingénierie de la santé 16, Bd Daviers, 49045, Angers, France AB-NM A.baumannii SAN A. baumannii PA O11 P. aeruginosa ATCC 33358, Liu PV, etal. Int. J. Syst. Bacteriol. 33: 256-264, 1983 DH5alfa E. coliInvitrogen BL- E. coli Novagen 21(DE3) NCBI: National Center forBiotechnology Information; ATCC: American Tissue Culture Collection,Virginia, USA1.1.3 A. baumannii reference genomes

Several published genomes were used for identification andcharacterization of identified targets as summarized in Table 7.

TABLE 7 Genome Sequences A. baumannii Genome References sequenceATCC19606 ¹NZ_ACQB00000000 AYE ¹NC_010410 ACICU ¹NC_010611 SDF¹NC_010400 AB307-0294 ¹NC_011595 6014059 ¹NZ_ACYS00000000 6013113¹NZ_ACYR00000000 6013150 ¹NZ_ACYQ00000000 AB0057 ¹NC_011586 ATCC 17978¹NC_009085 AB059 ¹NZ_ADHB00000000 AB058 ¹NZ_ADHA00000000 AB056¹NZ_ADGZ00000000 AB900 ¹NZ_ABXK00000000¹http://www.ncbi.nlm.nih.gov/genome

1.1.3 Patient Sera

Patient sera were collected in various hospitals. Sera form 20 patientswere described in previous studies (Pantophlet, R. et al. Clin. Diagn.Lab. Immunol. 7 (2), 293-295, (2000)) and were received from Prof.Seifert (Institute of Medical Microbiology and Hygiene, University ofCologne, Germany).

Further 57 patient sera were collected from hospitals in Athens(Greece), Sevilla (Spain), Pittsburgh (PE, USA) and Jerusalem (Israel).The following inclusion criteria were applied:

1. the patients have a confirmed A. baumannii bloodstream infections,pneumonia or severe wound infection,

2. the patient health status allows for blood collection and

3. that the patient is an adult less than 85 years of age. Patients withconfirmed viral infection (e.g. Hepatitis A, B or C, HIV), anemia or asuppressed immune system were excluded. All patients signed an informedconsent. Sera from healthy donors were collected from theSwiss-Red-Cross blood donation centre in Bern (Switzerland).

1.2 Approaches to Identify Suitable Targets 1.2.1 “Shedome” Analysis

The concept of this method is to identify polypeptides on theAcinetobacter membrane as they are accessible to large molecules such asantibodies. Thus live A. baumannii bacteria were shed with trypsin, a 23kDa protease, and analyzed by mass spectrometry (MS). The identifiedpeptides were assigned to proteins using public available databases. Itcan be expected that, besides contaminants of highly abundant proteinsand lysed bacteria, the digest contains peptides derived from proteinspresent on the extracellular side of the bacterial membrane.

1.2.1.1 Preparation of Bacterial Cultures

A. baumannii strain ATCC19606 was streaked onto an LBA plate andincubated overnight (16 h-24 h) at 37° C. The LBA plate with visiblebacterial colonies was kept at 4° C. for up to 1 month. As startingculture, 25 ml LB were inoculated using A. baumannii colonies from theLBA plate and incubated overnight at 37° C. shaking at 200 rotations perminute (rpm). The optical density at 600 nm (OD₆₀₀) of the overnightculture was measured. LB (0.4 l) was inoculated with overnight cultureat a starting OD₆₀₀ of 0.05 and incubated at 37° C. shaking at 200 rpmfor 3.5 h until an OD₆₀₀ of 0.68 was reached.

1.2.1.2 Trypsin Digests of Live Bacteria

Rodriguez-Ortega et al. (Nature Biotechnology, 24, 191-197, 2006)previously described a method for tryptic digest of gram positivebacteria, which was used to establish the following protocol for thegram negative A. baumannii. The bacteria were centrifuged at 3500 g for10 minutes at 4° C. The pellet was washed 3 times in 40 ml PBS (8% (w/v)NaCl, 2% (w/v) KCl, 1.1% (w/v) Na₂HPO₄, 0.2% (w/v) KH₂PO₄, pH=7.4) at 4°C. by resuspension and centrifugation. The pellet was washed once in 2ml sucrose buffer (PBS containing 40% (w/v) sucrose, 5 mM DTT(Dithiothreitol) and finally the pellet was resuspended in 2 ml sucrosebuffer containing 20 μg sequencing grade trypsin (Promega, V5113). Thesuspension was incubated for 30 minutes at 37° C. and then centrifugedfor 10 minutes at 3500 rcf at 4° C. The supernatant was removed andcentrifuged again for 5 minutes at 14000 rcf at 4° C. Again thesupernatant was removed and filtered through a sterile filter forsyringes (0.2 μm, Nalgene #194-2520). To 0.75 ml filtrate 0.75 μl formicacid were added, mixed and stored at −70° C. until analyzed by MS.

1.2.1.3 MS-Analysis of Tryptic Digest

Peptides were identified by mass spectrometry (nano LC-MS/MS withdata-dependent collision induced fragmentation) at the Department ofClinical Research, University of Berne, Switzerland by the group of Dr.Manfred Heller. The UniprotKB Database (The UniProt Consortium, NucleicAcids Res. 39: D214-D219, 2011), without entries from Firmicutes and E.coli, was used to assign peptides to proteins.

Briefly, a volume of 3 μl or 6 μl was loaded onto a pre-column (MagicC18, 5 um, 300 Å, 0.15 mm i.d.×30 mm length) at a flow rate of ˜5 μl/minwith solvent A (0.1% formic acid in water/acetonitrile 98:2). Afterloading, peptides were eluted in backflush mode onto the analyticalnano-column (Magic C18, 5 μm, 100 Å, 0.075 mm i.d.×75 mm length) usingan acetonitrile gradient of 5% to 40% solvent B (0.1% formic acid inwater/acetonitrile 4.9:95) in 60 min at a flow rate of ˜400 nl/min. Thecolumn effluent was directly coupled to an LTQ-orbitrap XL massspectrometer (Thermo Fisher Scientific, MA, USA,) via a nanospray ESIsource operated at 1.700 kV. Data acquisition was made in data dependentmode with precursor ion scans recorded in the Fourier transform detector(FT) with resolution of 60,000 (@ m/z=400) parallel to five fragmentspectra (CID) of the most intense precursor ions in the linear iontrap.CID mode settings were: Wideband activation on; precursor ion selectionbetween m/z range 360-1400; intensity threshold at 500; precursorsexcluded for 15 sec. CID spectra interpretation was performed withPHENYX on a local, dual quad core processor server run under Linux usingUniprotKB SwissProt and TrEMBL databases. Allowed, variablemodifications were: Met oxidation (limited to 2), Asn/Gln deamidation(2), and pyrrolidone carboxylic acid on N-terminal Glu (1). Parent andfragment mass tolerances were set to 20 ppm and 0.5 Da, respectively.Protein identifications were accepted as true positive if at least twodifferent peptides, resulting in a protein score of ≥10.0, wereidentified.

1.2.1.4. Data Analysis and Target Selection

Several identified proteins were intracellular proteins of highlyabundant proteins such as ribosomal proteins. To discriminate betweensuch contaminants and putative membrane targets, the identified proteinswere analyzed for their localization within the bacteria using publiclyavailable online tools.(http://bp.nuap.nagoya-u.ac.jp/sosui/sosuigramn/sosuigramn_submit.html,K. Imai et al., Bioinformation 2(9), 417-421, 2008). Proteins that wereassigned as extracellular or outer membrane protein were selected forfurther analysis. In addition, proteins that were annotated by theUniprotKB Database as a homologue to known extracellular or outermembrane proteins were selected as well.

1.2.2. Comparative Proteomics

The concept of this method is to focus on polypeptides for whichexpression is experimentally confirmed in various and differentAcinetobacter strains. Accordingly, the whole proteome of five A.baumannii strains was determined by mass spectrometry. The five strainswere selected due to their diverse sources of isolation. To enrich forputative targets that are present on the extracellular surface, proteinpreparations were enriched for outer membrane proteins prior MS analysisaccording to their hydrophilic and hydrophobic properties. The peptidesidentified by mass spectrometry were assigned to proteins using publiclyavailable databases and selected according to IT-predictions andliterature searches.

1.2.2.1 Preparation of Bacterial Cultures

A. baumannii strains ATCC19606, BMBF65, SDF, ACICU, AYE (see Table 6,above) were streaked onto BHI-Agar plates and incubated overnight (16h-24 h) at 37° C. The agar plates, containing visible bacterial colonieswere used to inoculate 75 ml LB and cultures were incubated for 25 h at37° C. shaking at 200 rpm. The OD₆₀₀ of the cultures was measured and LB(0.51) was inoculated with overnight culture at a starting OD₆₀₀ of0.02. The 0.5 l cultures were incubated overnight at 37° C. shaking at200 rpm. OD₆₀₀ was measured and 900 OD/ml of each culture were used forprotein preparation.

1.2.2.2 Outer Membrane (OM) Protein Preparations

OM-proteins were essentially prepared as described previously by Arnoldand Linke (Curr Protoc Protein Sci.; Chapter 4: Unit 4.8.1-4.8.30, 2008)with slight modifications to prepare OM-proteins for further downstreamanalysis. 900 OD/ml were pelleted at 4° C. for 20 minutes and 4000 g.All following steps were performed on ice with chilled solutions andapparatus at 0° C. to 4° C. The bacteria were resuspended in 7 mlresuspension buffer (0.1 M NaCl, 10 mM MgCl₂, 50 mM Tris-HCl, pH=8.0, 10mg/l DNase I (Sigma-Aldrich,)) and 0.1 ml protease inhibitor cocktail(Sigma-Aldrich,) was added. The suspension was sonicated 5 times for 10seconds at the level 5 using the Sonifier B-12 (Branson Sonic PowerCompany, CT, USA) with intervals of 1 minute on ice. The lysate wasincubated on ice for 30 minutes and subsequently centrifuged at 2000 gfor 15 minutes to remove intact bacteria. The supernatant wastransferred to centrifuge tubes, capable for ultracentrifugation, andresuspension buffer was added to a final volume of 12 ml. The solutionwas centrifuged at 100,000 g and 4° C. for 1 hour. Supernatant wasdiscarded and the pellet resuspended in 12 ml resuspension buffercontaining 0.1 ml protease inhibitor cocktail. The ultracentrifugationwas repeated and the pellet resuspended in 12 ml CM buffer (0.1 M NaCl,50 mM Tris-HCl, pH=8.0, 1% (w/v) Sodium N-Lauroylsarcosinate (Fluka)).0.1 ml protease inhibitor cocktail was added to the suspension and themixture incubated at room temperature for 30 minutes by rotating thetube on an intelli-mixer (LTF Labortechnik, Germany) set to an angle of90° and 25 rotations per minute. The solution was ultracentrifuged andthe pellet washed three times in 12 ml cold ddH₂O by resuspension andultracentrifugation as described above. At this stage the pellet wasfrozen at −20° C. until further use. The OM-protein preparation waschloroform/methanol precipitated (Wessel D. and Flügge U., Anal.Biochem. 138, 141-143, 1984) dividing the OM-protein preparation intotwo aliquots containing 45% and one aliquot containing the remaining10%. The pellets were stored at −20° C. For protein quantification thechloroform/methanol precipitated 10% aliquot was resuspended in 0.1 mlwater of which 50 μl were hydrolyzed with 50 μl 1M NaOH for 2 minutes atroom temperature and neutralized with 0.1 ml 0.5 M HCl. The hydrolyzedsample was titrated and protein quantified using Bradford proteinreagent (Biorad, CA; USA) according to manufacturer's instructions.Titrated bovine serum albumin, hydrolyzed like the samples, was used asa standard for quantification.

1.2.2.3 OM-Proteome Determination—LC-MS and Data Analysis

The proteins were solubilized in 8 M urea solution, reduced with 1 mMDTT for 30 mM at 37° C. and alkylated with 55 mM iodoacetamide for 30min in the dark at 25° C. The samples were then diluted with 0.1 Mammoniumbicarbonate buffer to a final urea concentration of 1 M.Proteins were digested by incubation with sequencing-grade modifiedtrypsin (1/100; w/w, Promega, Madison, Wis.) overnight at 37° C.Peptides were desalted on C18 reversed-phase spin columns according tothe manufacturer's instructions (Microspin, Harvard Apparatus), driedunder vacuum and stored at −80° C. until further use.

Peptide mixtures were analyzed using high-resolution nano-LC-MS on ahybrid mass spectrometer consisting of a linear quadrupole ion-trap andan Orbitrap (LTQ-Orbitrap XL, Thermo Fisher Scientific). Peptides wereanalyzed twice on an Eksigent Nano LC system (Eksigent Technologies)connected to a hybrid mass spectrometer consisting of a linearquadrupole ion-trap and an Orbitrap (LTQ-Orbitrap XL, Thermo FisherScientific), which was equipped with a nanoelectrospray ion source(Thermo Scientific). Peptide separation was carried out on a RP-HPLCcolumn (75 μm inner diameter and 10 cm length) packed in-house with C18resin (Magic C18 AQ 3 μm; Michrom Bioresources) using a linear gradientfrom 95% solvent A (water, 0.1% formic acid, and 2% acetonitrile) and 5%solvent B (water, 0.1% formic acid, and 98% acetonitrile) to 72% solventA and 28% solvent B over 60 min at a flow rate of 0.3 μl/min. TheLTQ-Orbitrap was operated in data-dependent acquisition mode with theXcalibur software. Survey scan MS spectra were acquired in the Orbitrapon the 350-2000 m/z range with the resolution set to a value of 60,000.The five most intense ions per survey scan were selected for collisioninduced dissociation (CID) fragmentation, and the resulting fragmentswere analyzed in the linear trap (LTQ). Dynamic exclusion was usedwithin 30 s to prevent repetitive selection of the same peptide. Singlycharged ions and ions with unassigned charge states were excluded fromtriggering MS/MS scans.

Raw data files from the MS instruments were converted with ReAdW intomzXML files and mzXML files were searched with Sorcerer-SEQUEST (Eng etal., J Am Soc Mass Spectrom. 1994; 5(11): 976-989) against aAcinetobacter baumannii protein database (ACIB3) from theUniProtKB/Swiss-Prot Protein Knowledgebase (Version 56.9) containing3453 protein entries (292 in UniProtKB/Swiss-Prot+3161 inUniProtKB/TrEMBL). Statistical analysis of each search result for eachLC-MS analysis was performed using the Trans-Proteomic Pipeline TPP(Keller et al., Mol Syst Biol. 2005; 1:2005.0017): v4.0 JETSTREAM rev 2including PeptideProphet (Keller A, et al., Anal. Chem. 2002; 74(20):5383-5392) and ProteinProphet (Nesvizhskii et al., Anal. Chem. 2003;75(17):4646-4658). The ProteinProphet probability score was set to 0.9,which resulted in an average protein and peptide false discovery rate ofless than 1% for all search results estimated by ProteinProphet andPeptideProphet.

The database search criteria included: 50 ppm mass tolerance for theprecursor ion, variable modifications of 15.994920 Da for methionines(representing oxidized methionines), 57.021465 Da forcarbamidomethylation as static modification for cysteines, at least onetryptic terminus per peptide, and up to two missed cleavage sites.

1.2.2.4 Data Analysis and Target Selection

To select for putative targets from the OM-proteome, the identifiedproteins of 5 different strains (see above, Table 6) were analyzed fortheir localization within the bacteria using publicly available onlinetools (PSORTb v3.0, Yu et al., Bioinformatics 26(13):1608-1615, 2010).Proteins that were present in the OM-proteome of all 5 strains andpredicted to locate either extracellular or to the outer membrane wereindividually analyzed in detail. This included the genomic conversationamong 14 publicly available reference genomes (presence/absence of geneand percentage of amino acid identity) and the predicted topology of theprotein within the outer membrane using the publicly available onlinetool HHpred (Söding et al., Nucleic Acids Res. 2005 Jul. 1; 33 (WebServer issue): W244-8.). If available literature concerning theAcinetobacter protein identified or homologues in other species wasconsidered as well.

Proteins that (1) were encoded by at least 13 of 14 genomes analyzedwith an amino acid conversation of ≥90% and (2) were predicted todisplay parts of the protein sequence on the extracellular side of theouter membrane were considered as putative antibody targets. In thosecases where the literature predicted homologues of such a putativeantibody target to be down-regulated or absent in antibiotic resistantA. baumannii strains, the targets were no longer followed. For instance,the outer membrane protein CarO was previously shown to bedown-regulated in Carbapenem resistant A. baumannii strains (Mussi etal., Antimicrob Agents Chemother. April; 49(4): 1432-40, 2005). Despitethe fact that the target was identified by comparative proteomics aswell as specific target selection, CarO was considered a target oflittle clinical relevance and therefore was not investigated further.

1.2.3. Specific Target Identification

This method focuses on specific targets that are recognized byantibodies present in sera of convalescent A. baumannii patients.Accordingly, OM protein preparations enriched for outer membraneproteins, were separated by 2-dimensional gel electrophoresis (2DE). The2DE consisted of an isoelectric focusing (IEF) followed bySDS-polyacrylamide gel electrophoresis (PAGE) step to resolve the OMproteins. Proteins recognized by patient sera were determined byimmunoblot analysis. To increase the chance of identifying proteins thatare expressed by various strains, immunoblots of at least two A.baumannii strains were compared and proteins present in all strainsanalyzed were selected for protein identification by MS-analysis. Theproteins were individually characterized and selected according toIT-predictions and literature searches.

1.2.3.1 Preparation of Bacterial Cultures and OM-Protein Preparations

A. baumannii strains ATCC19606, BMBF-65 and Berlin-95 (see Table 6) wereused to generate OM-protein preparation as described in 1.2.2.2.

1.2.3.2 Two-Dimensional Gel Electrophoresis (2DE)

Isoelectric focusing (IEF) was performed according to the manufacturer'sinstructions (GE Healthcare, United Kingdom) using the Ettan™ IPGphor™ 3IEF System (GE Healthcare). Briefly, Immobiline pH3-10 NL 7 cm DryStrips(GE Healthcare) were rehydrated overnight at room temperature in 125 μlrehydration solution (8M Urea (Sigma-Aldrich), 2% CHAPS (Sigma-Aldrich),40 mM DTT (Fluka,), 0.5% IPG buffer (GE Healthcare), 0.002% bromophenolblue). OM-preparations (20-30 μg) were dissolved in 50-100 μlrehydration solution, vortexed for 30 seconds and incubated at roomtemperature for several minutes. The sample was then centrifuged for 2minutes at >14000 g and the supernatant was used for IEF. In duplicates,sample was loaded onto rehydrated Immobiline DryStrips using thecup-loading system and overlaid with mineral oil. Proteins wereseparated using the running conditions 300V for 1 h, linear gradient300V-1000V for 30 minutes, linear gradient 1000V-5000V for 1 h 30minutes and 5000V for 36 minutes. The strips were frozen immediately at−20° C.

The 2^(nd) dimension was performed exactly as described by themanufacturer's instructions (Invitrogen, USA) using NuPAGE® Novex 4-12%Bis-Tris ZOOM® Gels (Invitrogen) and 10 μl of Novex® Sharp Pre-stainedProtein Standard (Invitrogen). One duplicate of the gels was used forblotting onto nitrocellulose membranes (Invitrogen) as described by themanufacturer's instructions for Tris-Glycine gels (Invitrogen), using30V for 80 minutes as running conditions. The nitrocellulose membranewas stained with Ponceau S solution (Sigma-Aldrich) and a picture wasrecorded. The membrane was incubated for 1 h at room temperature inblocking buffer (5% Skim milk (Fluka) in PBS-T (PBS containing 0.05%Tween 20® (Sigma-Aldrich). Individual patient sera or mixtures werediluted 1:500 in blocking buffer and incubated with the membraneovernight at 4° C. The membrane was washed three times for five minutesin PBS-T and incubated with a human IgG specific secondary antibody(Invitrogen) at a dilution of 1:1000 in blocking buffer for 1 h at roomtemperature. The membrane was washed again three times and boundantibody was detected using TMB substrate (Promega). Proteins that weredetected by a given patient serum in all A. baumannii strains tested,were selected for protein identification. Therefore proteins in thesecond duplicate of the 2DE-gels were visualized with Instant Blue™(Expedeon, Cambridgeshire, UK). Proteins positive in immunoblots werelocalized in the gel duplicate by comparing the protein pattern of thegel with the protein pattern of the Ponceau S stained membrane and theimmunoblot signals. The protein spots were excised and stored at −80° C.until protein identification by MS analysis.

1.2.3.3. MS-Analysis of Tryptic Digest

Proteins were identified from excised gel fragments by LC/ESI/MS/MS bythe Protein Analysis Group, Functional Genomics Center Zurich,Switzerland using standard procedure. Briefly, gel pieces were washedtwice with 100 μl 100 mM NH₄HCO₃/50% acetonitrile, washed with 50 □lacetonitrile. All three supernatants were discarded and 10 μl trypsin(100 ng in 10 mM Tris/2 mM CaCl₂, pH 8.2), 20 μl buffer (10 mM Tris/2 mMCaCl₂, pH 8.2) added and incubated overnight at 37° C. Supernatant wasremoved and gel pieces extracted twice with 100 μl 0.1% TFA/50%acetonitrile. All three supernatants were combined and dried. The samplewas dissolved in 25 μl 0.1% formic acid and transferred to anautosampler vial for LC/MS/MS. 5 μl were then injected for peptideidentification. Database searches were performed by using theProteinLynx Global Server (SwissProt, all species) and Mascot (NCBInr,all species) search programs.

1.2.3.4. Data Analysis and Target Selection.

Proteins that were identified as A. baumannii protein and predicted tobe or annotated as an outer membrane protein were chosen as putativetargets. The targets were no more followed in cases where the literaturepredicted homologues of such a putative antibody target to bedown-regulated or absent in antibiotic resistant A. baumannii strains.

Example 2: IT Predictions

For IT predictions of protein structure the Bioinformatics Toolkit formthe Max-Planck Institute for developmental Biology in Tubingen was used(Biegert et al., Nucleic Acids Res. 34, W335-339). Tertiary structureswere predicted using the tool HHpred (Söding J. et al., Bioinformatics,2005, 21, 951-960,) that builds a hidden Markov Model (HMM) of the querysequence and compares it with a database of HMMs, representing annotatedprotein families (e.g. PFAM, SMART, CDD, COGs, KOGs) or domains withknown structure (PDB, SCOP). As a setting for the online prediction, theHMM database pdb70_3 Sep11 was used and HHblits were set to MSAgeneration method with maximal 3 iterations and local alignment mode.The predicted structures were assumed to be true with a high probabilityto be a true positive (>90%) and a homology that covers most of thequery sequence. Where multiple hits met the requirements, the two hitswith the highest probability and lowest E- and P-values were used asrepresenting tertiary structure. Representing predicted tertiarystructures to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 are assigned inTable 4 and can be downloaded from the pubmed online server or otherwiseobtained with reference to Wang Y. et al., Nucleic Acids Res. 2007January.

Prediction of N-Terminal Leader Sequence:

The N-terminal leader sequence was determined using the SignalP 3.0Server, Bendtsen J. D. et al., J. Mol. Biol., 340:783-795, 2004.) forgram negative bacteria using hidden Markov models and Neural Networks.

Prediction of Sub Cellular Protein Localization:

The public available online tool Psortb v3.0 Yu et al., 2010,Bioinformatics 26(13):1608-1615) was used for prediction of sub cellularlocalization. “Bacteria” and “gram-negative stain” were chosen assettings for prediction. The protein sequences were entered as singleletter amino acid code.

For the shedome analysis, the public available online tool SOSUI_(GramN)was used Imai et al., Bioinfomation 2(9), 417-421 (2008), entering theprotein sequences as single letter amino acid code.

Determination of Amino Acid Conservation and Gene Prevalence:

For determination of gene prevalence and amino acid conservation, theamino acid sequence to be analyzed was entered into the genomic blastonline tool “tblastn” (Cummings L, et al., FEMS Microbiol Lett. 2002Nov. 5; 216(2):133-8; Altschul et al., Nucl Acid Res., 25:3389-3402,(1997)) as query sequence using single amino acid single letter code.All A. baumannii genomic databases were selected. Default BlastPparameters were chosen (BLOSUM62 Matrix, Gap costs to open=11, Gap coststo extend=1, using low complexity filter and composition basedstatistics). The Expect-values to be accepted was kept at a defaultsetting of 10. Of the results, the prevalence and the percentage aminoacid identity among the reference genomes (Table 4) was used for targetselection.

In cases where a DNA sequence was used as a Query sequence, blastn wasused instead, using default settings (BLOSUM62 Matrix, Gap costs toopen=5, Gap costs to extend=2, Match scores=2, mismatch score=−3).Depending on the length of the sequence the program used low or strongcomplexity filter.

Example 3: Generation of Expression Vectors for the Generation ofRecombinant Antigens

The nucleic acid sequences encoding the polypeptides of the presentinvention were amplified by PCR from genomic DNA of A. baumannii(ATCC19606) using primers containing appropriate restriction sites forcloning. PCR products were cloned in frame into the expression vectorpET-28a(+) (Novagen; Germany) resulting in recombinant protein with anN-terminal His-tag. All oligonucleotides were generated at Microsynth(Balgach, Switzerland). For AB023, AB024, AB025, AB030, FimA, CsuAB andOmpA the whole coding sequence (cds) without the N-terminal signalpeptide was cloned. The N-terminal leader sequence was determined andremoved for cloning and expression of recombinant proteins. For AB031the 78 amino acid extracellular loop was cloned as it was the onlyregion of more than 2 amino acids within this molecule predicted to beon the extracellular side and therefore accessible to antibodies. Theexpression plasmids were sequenced at Microsynth to exclude PCRartifacts. SEQ ID NOs: 33 and 34 show the nucleotide sequence of thesequencing primer T7 and T7 term respectively. An additional sequencingprimer, consisting of the nucleotide sequence described as SEQ ID NO:35, was used for the expression vector of AB030 described in example3.4.

3.1 Expression Vector for AB023 (SEQ ID NO: 1)

SignalP 3.0 Server predicted an N-terminal signal sequence at position1-26 for SEQ ID NO: 2. The oligonucleotides oAB023wss GGCAGGATCCGCTGCTGCATTTGACCC (SEQ ID NO: 17) and oAB023as CGGAATGTCGACTTAGAA TGCAGTTG(SEQ ID NO: 18) were designed to bind at the position 76-95 and1241-1254 of SEQ ID NO: 1 respectively. Restriction sites, added to theoligonucleotides oAB023wss and oAB023as for cloning, are underlined. Thecds homologues to SEQ ID NO: 1 position 76-1254 was amplified by PCRfrom genomic DNA of ATCC19606 using the P:lX Polymerase (Invitrogen) andthe oligonucleotide pair oAB023wss/oAB023as. Per 50 ul reaction, 50 ngof genomic DNA, 1 U P:lX polymerase, 1 mM MgSO4, 2×pfx buffer, 0.3 mMdNTP (each), 0.3 flM oligonucleotide (each) were used. The PCR thermocycle program was (94° C., 4 min) 35× (94° C., 15 s; 55° C., 30 s; 65°C., 2 min) (65° C., 5 min). The PCR product was purified using QIAquickgel extraction kit (QIAGEN, 28704) according to the manufacturesinstructions. The purified PCR product and 100 ng of the vectorpET-28a(+) were digested using the restriction enzymes BamHI and SalI(Fermetas, ER0051, ER0641) and the digests were purified by usingQIAquick PCR Purification Kit (QIAGEN, 28104) according to themanufactures instructions. Subsequently 50 ng vector was ligated at amolar ratio of 1:2 with the PCR product for 2 h at room temperatureusing 2 units ligase (Fermentas, Canada) in a total volume of 20 ul andIx ligase buffer (supplied with ligase). Ligation reaction wastransformed into chemicompetent E. coli (DH5a) and selected onLBA-plates containing 50 ug/rnl kanamycin (Applicem) using standardprocedures (Maniatis). Resistant colonies were selected for purificationof plasmid DNA using commercially available kits (Promega, Wis., USA orQIAGEN, (Germany) and purified plasmids were sequenced at Microsynth(Balgach, Switzerland) using the standard sequencing primers T7(TAATACGACTCACTATAGG—SEQ ID NO: 33) and T7 term(TGCTAGTTATTGCTCAGCGG—SEQ ID NO: 34) to verify correct integration ofthe PCR product. The expression vector for AB023 encoded the same aminoacid sequence as expected from the Acinetobacter genome sequence ofATCCI9606 (DOCDE3) except for the signal peptide (amino acids 1-26) thatwas replaced by the His-tag from the vector. 3.2 Expression vector forAB024 (SEQ ID NO: 3)

SignalP 3.0 Server predicted an N-terminal signal sequence at position1-29 for SEQ ID NO: 4. The oligonucleotides oAB024wssGGCAGGATCCGCAACTTCTGATAAAGAG (SEQ ID NO: 19) and oAB024asCAAAGTCGACTTAGAAGCTATATTTAGCC (SEQ ID NO: 20) were designed to bind atthe position 88-105 and 1287-1305 of SEQ ID NO: 3 respectively.Restriction sites, added to the oligonucleotides oAB024wss and oAB024asfor cloning, are underlined. The cds homologues to SEQ ID NO: 3 position88-1305 was amplified by PCR and cloned into pET-28a(+) exactly asdescribed for the expression vector of AB023.

The expression vector for AB024 encoded the same amino acid sequence asexpected from the Acinetobacter genome sequence of ATCC19606 (D0CDN5)except for the signal peptide (amino acids 1-29) that was replaced bythe His-tag from the vector.

3.3 Expression Vector for AB025 (SEQ ID NO: 5)

SignalP 3.0 Server predicted an N-terminal signal sequence at position1-21 for SEQ ID NO: 6. The oligonucleotides oAB025wssTCGCGGATCCCAAGGTTTAGTGCTTAATAATGATG (SEQ ID NO: 21) and oAB025asCGACAAGCTTAGAAACCAAACATTTTACGCTC (SEQ ID NO: 22) were designed to bindat the positions 67-88 and 1422-1446 of SEQ ID NO: 5 respectively.Restriction sites, added to the oligonucleotides oAB025wss and oAB025asfor cloning, are underlined. The cds homologues to seq5 position 67-1446were amplified by PCR and cloned into pET-28a(+) exactly as describedfor the expression vector of AB023 with the modification that therestriction enzyme HindIII (Fermentas, ER0501) was used instead of SalI.

The expression vector for AB025 encoded the same amino acid sequence asexpected from the Acinetobacter genome sequence of ATCC19606 (D0C8X7)except for the signal peptide (amino acids 1-21) that was replaced bythe His-tag from the vector.

3.4 Expression Vector for AB030 (SEQ ID NO: 7)

SignalP 3.0 Server predicted an N-terminal signal sequence at position1-44 for SEQ ID NO: 8. The oligonucleotides oAB030wssCTTGTGGATCCCAAAGTTCGGCTGAGACC (SEQ ID NO: 23) and oAB030asAAAGTCGACTTAAAGTTGTGGACCAATAAAGAAATG (SEQ ID NO: 24) were designed tobind at the position 133-150 and 2695-2721 of SEQ ID NO: 7 respectively.Restriction sites, added to the oligonucleotides oAB030wss and oAB030asfor cloning, are underlined. The cds homologues to SEQ ID NO: 7 position133-2721 were amplified by PCR and cloned into pET-28a(+) exactly asdescribed for the expression vector of AB023 with the modification thatthe elongation time of the PCR was increased to 2 min 30 sec and thecycle number reduced to 30.

The expression vector for AB030 encoded the same amino acid sequence asexpected from the Acinetobacter genome sequence of ATCC19606 (D00629)except for the signal peptide (amino acids 1-44) and the amino acid atposition 58 that encodes for a threonine instead of serine. Sincehomologues of AB030 in other Acinetobacter baumannii strains (e.g.AB307-B7H123) contain at this position a threonine, this difference fromthe expected sequence was tolerated.

3.5 Expression Vector for AB031L (SEQ ID NO: 9)

The homology detection and structure prediction software HHPred (Södinget al., Nucleic Acids Res.; 33(Web Server issue):W244-8, 2005 Jul. 1)was used to predict the structure of AB031. A structural homologue ofAB031 (Pubmed Protein ID lek9—Outer membrane protein TOLC) was predictedwith highest probability (100%) and an E-value (0) of higheststatistical significance. The alignment predicted the 78 amino acidsequence at position 87-164 of SEQ ID NO: 10 to locate to theextracellular side of the bacteria.

The oligonucleotides oAB031L1wss AAAGGATCCAGAGCATATGCTTTTCATAGTG (SEQ IDNO: 25) and oAB031L1as AAAGTCGACTTAAGATGGTCGGACTACTTGGTCTTCT (SEQ ID NO:26) were designed to amplify the 78 amino acid loop by PCR. Restrictionsites, added to the oligonucleotides oAB031L1ss and oAB031L1as forcloning, are underlined. The cds homologues of the 78 amino acidsequence was amplified by PCR from genomic DNA of ATCC19606 using theDream-Taq polymerase (Fermentas, EP0701) and the oligonucleotide pairoAB031L1wss/oAB031L1as. Per 50 μl reaction, 50 ng of genomic DNA, 0.5 Utaq polymerase, 1×taq buffer, 0.2 mM dNTP (each), 0.2 μM oligonucleotide(each) were used. The PCR thermo cycle program was (94° C., 3 min) 5×(94° C., 15 s; 50° C., 15 s; 72° C., 2 min) 25× (94° C., 15 s; 55° C.,15 s; 72° C., 2 min) (72° C., 5 min). The PCR product was cloned intopET-28a(+) as described for the expression vector of AB023. Theexpression vector for AB031L1 encoded the same amino acid sequence asexpected from the 78 amino acid sequence of SEQ ID NO: 10.

3.6 Expression Vector for FimA (SEQ ID NO: 11)

SignalP 3.0 Server predicted an N-terminal signal sequence at position1-20 for SEQ ID NO: 12. The oligonucleotides oFimAwssGGACGAGGATCCGCTGATGGTACAATTACA (SEQ ID NO: 27) and oFimAasAACTAAGCTTTCAACCCATTGATTGAGCAC (SEQ ID NO: 28) were designed to bind atthe position 61-78 and 392-407 of SEQ ID NO: 12 respectively.Restriction sites, added to the oligonucleotides for cloning, areunderlined. The cds homologues to seq11 position 61-407 was amplified byPCR and cloned into pET-28a(+) exactly as described for the expressionvector of AB025.

The expression vector for FimA encoded the same amino acid sequence asexpected from the Acinetobacter genome sequence of ATCC19606 (D00767)except for the signal peptide (amino acids 1-20) that was replaced bythe His-tag from the vector.

3.7 Expression Vector for CsuAB (SEQ ID NO: 13)

SignalP 3.0 Server predicted an N-terminal signal sequence at position1-23 for SEQ ID NO: 14. The oligonucleotides oCsuABwssAATACTGGATCCGCTGTTACTGGTCAG (SEQ ID NO: 29) and oCsuABasAACTAAGCTTTTAGAAATTTACAGTGACTAATAGAG (SEQ ID NO: 30) were designed tobind at the position 70-84 and 512-537 of SEQ ID NO: 13 respectively.Restriction sites, added to the oligonucleotides oCsuABwss and oCsuABasfor cloning, are underlined. The cds homologues to SEQ ID NO: 13position 70-537 was amplified by PCR and cloned into pET-28a(+) asdescribed for the expression vector of AB025.

The expression vector for CsuAB encoded the same amino acid sequence asexpected from the Acinetobacter genome sequence of ATCC19606 (D0C5S9)except for the signal peptide (amino acids 1-23) that was replaced bythe His-tag from the vector.

3.8 Expression Vector for OmpA (SEQ ID NO: 15)

SignalP 3.0 Server predicted an N-terminal signal sequence at position1-22 for SEQ ID NO: 16. The oligonucleotides oOmpAwssCTGCTGAATTCGGCGTAACAGTTACTCC (SEQ ID NO: 31) and oOmpAasCAAGAAAGCTTATTATTGAG (SEQ ID NO: 32) were designed to bind at theposition 67-83 and 1064-1071 of SEQ ID NO: 15 respectively. Restrictionsites, added to the oligonucleotides oOmpAwss and oOmpAas for cloning,are underlined. The cds homologues to SEQ ID NO: 15 position 67-1071were amplified by PCR and cloned into pET-28a(+) exactly as describedfor the expression vector of AB023 with the modification that therestriction enzymes EcoRI and HindIII (Fermentas, ER0271, ER0501) wereused instead.

The expression vector for OmpA encoded the same amino acid sequence asexpected from the Acinetobacter genome sequence of ATCC19606 (D0CDF2)except for the signal peptide (amino acids 1-22) that was replaced bythe added His-tag from the vector.

Example 4: Expression and Purification of Recombinant Proteins

4.1 Expression of Recombinant Proteins in E. coli.

For recombinant expression of His-tagged proteins, chemicompetent E.coli BL-21(DE3) were transformed with the individual expression vectorsdescribed above and selected on LBA-plates containing 50 μg/ml kanamycinusing standard procedures. Overnight culture in LB containing 50 μg/mlkanamycin of resistant colonies were used to start a 0.5 l LB culturecontaining 50 μg/ml kanamycin at an OD₆₀₀ of 0.2 or lower. The culturewas incubated at 37° C. and 200 rpm until an OD₆₀₀ of 0.5-1 was reached.IPTG (Sigma-Aldrich) was added at a concentration of 1 mM and bacteriawere incubated further at 37° C. and 200 rpm for 3-4 h. Bacteria werecentrifuged (3500 g, 10 min) and pellet was frozen at −20° C.

4.2 Extraction of Recombinant Proteins from E. coli Bacterial Pellets.

Bacterial cell pellet was resuspended in 10 ml cell disruption buffer(0.15 M NaCl, 10 mM MgCl₂, 10 mM MnCl₂, 20 mM Tris-HCl, pH=8.0, 10 mg/lDNaseI), the suspension was sonicated on ice as described in 1.2.2.2 andincubated on ice for 30 minutes. The suspension was centrifuged (4000 g,10 min at 4° C.), supernatant was discarded and pellet resuspended in 10ml detergent buffer (0.15 M NaCl, 20 mM Tris-HCl, pH=8.0, 1% TritonX100) by mechanical forces. The suspension was centrifuged at 8000 g, 4°C. for 10 minutes. In case of his tagged AB031L1, the supernatant wassupplemented with 5 mM DTT to generate AB031L1 binding buffer andimmediately used for Ni-NTA affinity purification. For all otherrecombinant proteins, the supernatant was discarded and the pellet waswashed twice in 20 ml deionized cold water by resuspending the pelletand repeating the centrifugation. The washed pellet was frozen at −20°C. until further use.

Recombinant protein was extracted in 10-20 ml binding buffer byincubating the resuspended pellet for 30 min rotating at roomtemperature. For His-tagged FimA, the pellet was extracted with bindingbuffer G (6M GuHCl, 0.5 M NaCl, 20 mM Imidazole (Merck, Germany), 5 mMDTT, 20 mM Tris-HCl, pH=9.0) while for His-tagged AB023, AB024, AB025,AB030, CsuAB and OmpA the pellet was extracted with binding buffer U (8M Urea, 0.5 M NaCl, 20 mM Imidazole, 5 mM DTT, 20 mM Tris-HCl, pH=8.0).

4.3 Ni-NTA Purification of Recombinant His Tagged Proteins.

HisTrap™ HP columns (GE Healthcare, 17-5247-01) were used for affinitypurification of his-tagged proteins. The Äkta avant apparatus (GEHealthcare) was used to operate the purification at a system flow rateof 1 ml/min and 0.5 MPa pre and 0.3 MPa delta column pressure limit. Thecolumns were equilibrated with 5 column volumes (CV) running buffer. Therunning buffer consisted of the same components as the binding bufferfor each antigen, except that no DTT was present. Binding buffercontaining the extracted recombinant proteins were applied to the columnand the column was washed with running buffer until the UV 280 nm signalrecorded was stable. Bound proteins were eluted from the column using 10CV of a linear gradient of 20 mM to 500 mM imidazole in running buffer.Fractions of 0.5 ml were collected and analyzed for presence, purity andquantity of recombinant protein by SDS-PAGE and Coomassie stainingrespectively. Fractions of highest purity and concentration ofrecombinant protein were pooled and quantified by comparison titratedrecombinant protein with a titrated BSA standard (0.5, 1, 2, 4, 6 μg perlane) on an SDS-PAGE gel stained with Coomassie.

FimA was precipitated by adding ethanol to 90% (v/v), cooled to −80° C.and centrifuged at >14,000 rcf at 4° C. for 30 minutes and dried bySpeed Vac. FimA was either stored as a pellet or dissolved in bindingbuffer U at a concentration of 1 mg/ml at −20° C. All other proteinswere diluted in running buffer to 1 mg/ml or 2 mg/ml and stored at −20°C.

4.4 Refolding of OmpA

OmpA was refolded according to McConnell et al. (McConnell, Michael J.;Pachón, Jerónimo (2011): Protein Expression and Purification 77 (1), S.98-103). Briefly, his tagged OmpA (1 ml at 1-2 mg/ml) was 50-folddiluted in 50 ml refolding buffer (10 mg/ml n-octyl-□-D-glucopyranoside,20 mM NaPi, pH 7.4) and incubated overnight at 42° C. The volume wasconcentrated to 1 mg/ml OmpA using Amicon Ultra-15 centrifugal deviceswith a 10 kDa cut off (Millipore, MA, USA).

Example 5: Generation of Polyclonal Rabbit Sera and Purification ofRabbit IgG

Antigens were individually prepared for generation of rabbit immunesera. AB030 was ethanol precipitated and resuspended in 1 M Urea buffer(1 M Urea, 10 mM Tris-HCl, pH=8.0, 0.1% SDS) at a concentration of 1.2mg/ml. AB031-L1 was precipitated and the pellet dissolved in 1 M Ureabuffer at a concentration of 2.5 mg/ml. Antigens (1.5 mg each) were sentto Biogenes (Berlin, Germany) where rabbit antisera were generated. Ofeach rabbit preimmune serum was taken before immunization. For eachantigen, two rabbits were immunized and boosted 7 and 14 days afterimmunization. On day 28, animals were boosted and 20 ml serum preparedand analyzed by ELISA and immunoblot analysis using recombinant protein.Total serum was prepared between day 42 and 56 after immunization. Seracontained 0.02% thimerosal as preservative.

Total IgG was purified from serum by protein A affinity purificationusing standard protocols. Purified total IgG was either in Tris-Glycinebuffer pH=7.5, 250 mM NaCl, 0.02% thimerosal or in Tris-Glycine bufferpH=7.5.

Thimerosal was removed by dialysis prior to experiments with livebacteria. Briefly, sera and total IgG were dialyzed twice for 30 minutesat room temperature and once overnight at 4° C. against 1-2 l PBS usingSlide-A-Lyzer dialysis cassettes with a 10 kDa cut off (ThermoFisherScientific, MA, USA).

Example 6: Immunoblot Analysis

Reference strains (E. coli, P. aeruginosa or A. baumannii) or clinicalisolates of A. baumannii were grown in LB media (if not otherwisementioned) to stationary phase or logarithmic phase (OD₆₀₀ 0.3-1.2) andcentrifuged for 5-10 min at 4000 g. Bacterial cell pellets wereresuspended in water and lysed with an equal volume of 2×SDS samplebuffer (0.1 M Tris-HCl pH=6.8, 4% (w/v) SDS, 0.2% (w/v) bromophenolblue, 20% glycerol, 0.2 M DTT) or 2× Novex® Tris-Glycine SDS SampleBuffer with reducing agent (LC2676, Invitrogen) at a final concentrationequivalent to 12 OD₆₀₀/ml and heated for 10 minutes at 98° C. Purifiedproteins were diluted in SDS sample buffer accordingly, reaching aconcentration of 1-2 μg per 10 μl or an equivalent OD₆₀₀/ml. Per lane ofa Novex® 4-20% Tris-Glycine gel (Invitrogen), 10 μl of bacterialsuspension or purified antigen were loaded. 5-10 μl molecular weightstandards (SeeBlue® Pre-stained, or Novex® Sharp Pre-stained ProteinStandard, Invitrogen) were loaded on a separate lane. Proteins wereseparated by SDS-PAGE, according to the manufacturer's instructions,using the running conditions 140 V for 90 minutes (Invitrogen). In caseswhere only purified antigens were separated, NuPAGE® 4%-20% Bis-Trisgels (NP0322BOX, Invitrogen) were used instead and separated accordingto the manufacturer's instructions for denatured, reduced samples usingMES running buffer (Invitrogen).

Gels were either stained with Coomassie as described above or blottedonto a nitrocellulose membrane and analyzed by Ponceau S staining andimmunoblot analysis as described above for 2DE. Rabbit antisera werediluted 1:500-1:1000 and human sera 1:500. Secondary antibodies,HRP-Goat anti-rabbit IgG (Sigma-Aldrich) and HRP-Goat anti-human IgG(Invitrogen), were used at a dilution of 1:2000.

Results of immunoblot analysis are shown in FIGS. 3, 4, 5, 6C and 9C.

Example 7: ELISA

96-well ELISA plates (Nun, 439454) were coated overnight at 4° C. or for2 h at room temperature for each antigenic polypeptide diluted incoating buffer at 1 ng/ml and 0.1 ml per well. Urea running buffer [8 Murea, 0.5 M NaCl, 20 mM Imidazol, 20 mM Tris-HCl, pH 8.0] was used forHis-tagged AB023, AB024, AB025, AB030, FimA and CsuAB as coating buffer.PBS was used as coating buffer for refolded OmpA and AB031 L1.

Coated ELISA plates were washed three times with PBS-T (0.35 ml per wellusing Skan washer 400, Skatran). Human sera or rabbit sera were used asprimary antibody. Primary antibody was diluted in PBS-T and 0.1 ml addedto each well. Prior to use as primary antibody human sera were titratedstarting at a dilution of 1:200 and rabbit antisera were titratedstarting at a dilution of 1:100 or 1:200. ELISA plates were incubatedwith primary antibodies for 1 h at room temperature and then washedthree times in PBS-T. HRP-Goat anti-human IgG (Invitrogen) or HRP-Goatanti-rabbit IgG (Sigma-Aldrich) were used as secondary antibodies at adilution of 1:2000 and 1:5000, respectively. ELISA plates were washedagain three times in PBS-T and bound HRP was detected by the colorchange of 0-Phenylenediamine (Fluka). The reaction was stopped using 1 MHCl and quantified by measuring the OD at 490 nm.

Use of human sera as primary antibody allows detection of targets whileuse of rabbit sera proves the immunogenicity of the targets.

Results of ELISA are shown in FIGS. 1 and 2.

Example 8: Bacterial FACS Analysis

OD₆₀₀ of stationary phase bacteria or logarithmic growing bacteria wasmeasured. Bacteria were diluted in PBS containing 0.5% (w/v) BSA asblocking agent at an OD₆₀₀ of 0.1. Per reaction 0.05 ml of bacterialsuspension was used and combined with 0.05 ml primary antibody in roundbottom 96-well cell culture dishes (Corning, N.Y., USA). Unboundantibody was removed by two washing cycles consisting resuspendedbacteria in 0.2 ml blocking agent, centrifugation for 10 minutes at 1700g and removal of supernatant. Optionally at this stage, bound antibodywas fixed by incubation in 4% (w/v) formaldehyde/PBS for 10 min on ice.If fixative was used, bacteria were washed twice. Secondary antibody,Goat anti-human IgG-Alexa Fluor 488, Goat anti-human IgM-Alexa Fluor 488or Goat anti-rabbit IgG-FITC (Invitrogen), 0.1 ml per well, were addedat a dilution of 1:1000 and incubated for 30 minutes. Bacteria werewashed again and analyzed using a FACS Calibur. Instrument at settingsadjusted to optimally discriminate the bacterial population from debrisand weak from strong fluorescent signals (Forward scatter: Voltage E01,Amp. Gain: 7.0, log. Sideward scatter: Voltage 659, Amp. Gain: 1.0, log,Fl-1: Voltage 767, Amp Gain: 1.0, log.). As negative control, washbuffer only, no primary antibody or preimmune serum were used. Patientsera or rabbit immune sera were used as positive a control (strongsignal). All solutions (except bacterial solutions) were sterilefiltered to reduce FACS artifacts.

Results are shown in FIGS. 6 A and B and in FIG. 7

Example 9: Immunofluorescence Analysis (IFA)

Various methods were used to prepare bacteria for IFA. Bacterialcolonies from LBA or BHI plates were resuspended in 50 μl water at highdensities (OD₆₀₀>1) and smeared onto a well of 10-well glass slides (MPBiomedicals Inc., USA). Liquid bacterial cultures were smeared directlyonto the slides. The smears were air dried and fixed for 10 min with 4%(w/v) formaldehyde in PBS followed by 3 washing steps using PBS.Alternatively bacteria were fixed for 10 min in −20° C. acetone and airdried. Another approach to prepare bacteria for IFA was to grow liquidbacterial cultures directly on glass slides (BD Biosciences, NJ, USA) toenable biofilm formation. Culture was removed and bacteria attached tothe glass slide were fixed as described above.

IFA was performed as follows: fixed bacteria were incubated withblocking agent (PBS containing 1% (w/v) BSA) for at least 30 minutes.Buffer was replaced by primary antibody diluted in blocking reagent.Rabbit immunsera were diluted 1:50-1:500. After incubation for 1 hourbacteria were washed 3-4 times with PBS. Secondary antibodies (goatanti-rabbit IgG-FITC (F2765, Invitrogen), diluted in blocking reagent ata dilution of 1:200-1:400, were incubated for 45 minutes and washed 3-4times with PBS. Slides were overlaid with Vectashield containing DAPI(H-1200, Vector labs) and sealed with a cover slide and nail varnish.Slides were analyzed and pictures taken using the 100-fold oil immersionobjective of the Nikon fluorescence microscope “fluonik” at theInstitute of Anatomy at the University of Berne, Switzerland. All stepswere performed at room temperature.

Results are shown in FIG. 8B

Example 10: Agglutination Assay

Stationary phase bacteria were diluted in PBS to an OD₆₀₀ of about 3.Logarithmic phase bacteria were concentrated by centrifugation andresuspension in PBS to an OD₆₀₀ of about 3. On a multiwall glass slide,100 □μl bacterial suspension was mixed with an equal volume of antibodyat a concentration of 0.2-1.5 mg/ml for total IgG purified from rabbitsera. The concentration depended on the characteristics of theindividual antibodies. Monoclonal and affinity purified polyclonalantibodies need a much lower concentration compared to total IgGpurified from immunsera. The slide was gently agitated and incubated atroom temperature for 10 minutes. Agglutination was observed using aMotic System Microscope (B1 Series) at a 10×-40× magnification.

Results are shown in FIG. 8A

Example 11: Direct FimA Pull Down Assay

A 20 μl bed volume of protein A beads (POROS® MabCapture™, AppliedBiosystems®, CA, USA) was washed twice in 1 ml PBS by centrifugation(300 g, 1 min) and removal of the supernatant. Beads were coated withantibody by incubating beads with 10 μg antibody in 0.2 ml PBS for 30minutes and 30 rpm at room temperature. Beads were washed again twice in1 ml PBS and beads were taken up in 0.4 ml supernatant of a LB overnightculture of A. baumannii. Supernatant was prepared by centrifugation ofthe bacterial culture at >4000 g for 5 minutes and supernatant wasfiltered through a 0.2 μm filter for syringes (Nalgene #194-2520). Themixture was incubated for 1 h and 30 rpm at room temperature. Beads werewashed again twice in 1 ml PBS. Finally, beads were resuspended in 30 μllysis buffer for NuPAGE® 4%-20% Bis-Tris gels (NP0322BOX, Invitrogen)and incubated at 98° C. for 5 min. The sample was tested for presence ofnative FimA by immunoblot analysis as described above according to themanufacturer's instructions for denatured, reduced 4%-20% Bis-Tris gelsusing MES running buffer (IM-8042 Version H, Invitrogen). Rabbit immuneserum against FimA was used for detection of FimA.

Results are shown in FIG. 11.

Example 12: Active and Passive Immunization in Animals

Active and passive immunization studies were performed using the mouseAcinetobacter pneumonia model using as read outs percentage survival,clinical scores and body weights previously developed by Eveillard etal., 2010, Journal of Infection 60 (2), S. 154-161.

12.1 Active Immunization

On days 0, 14, 28, 42, each mouse (135 C3H/HeN mice, 18-20 g, 6 weeksold. Elevage Janvier, Sarthe, France) was immunized intra peritonealwith 10 μg antigen in 0.1 ml 50% (v/v) Gerbu adjuvant (GERBU BiotechnikGmbH, Germany)/PBS. As negative controls, mice were either immunizedwith 50% (v/v) Gerbu adjuvant/PBS or PBS only.

On day 49, the pneumonia model was started according to the establishedprotocols at the laboratory of Marie Laure Joly-Guillou and MatthieuEveillard (Eveillard et al., Journal of Infection 60 (2), S. 154-161,2010). Briefly, the mice were rendered transiently neutropenic byinjecting cyclophosphamide (Baxter, Ill., USA) by intra-peritonealinjection (150 mg/kg body weight in 0.15 ml) on days 4 and 3 before A.baumannii inoculation. The mice were anesthetized by isoflurane inconjunction with pure oxygen. Intra-tracheal instillation of A.baumannii was performed as previously described (Joly-Guillou et al.,Antimicrob Agents Chemother. February; 41(2):345-51, 1997). Briefly, thetrachea was cannulated with a blunt needle and 50 μl of a bacterialsuspension containing 10⁸ cfu/mL were deposited. Inoculum size wasconfirmed by quantitative culture.

After intra-tracheal instillation of the inoculums, the mice werereturned to their cages (day 0) and observed to assess spontaneousoutcome. This outcome was evaluated daily (including day 0) andconcerned mortality, mouse weight changes, and a clinical score built onthe basis of mice mobility (score=0 for a spontaneous mobility, score=1when a mobility was only observed after stimulation, and score=2 for anabsence of mobility), the development of a conjunctivitis (score=0 inthe absence of conjunctivitis, score=1 when there was a conjunctivitis),and the aspect of hair (score=0 for a normal hair and score=1 forruffled hair). Overall, this clinical score varies from 0 for normalmice to 4 for severe illness.

Results are shown in FIG. 12.

12.2 Passive Immunization

The pneumonia model was started according to the established protocolsat the laboratory of Marie Laure Joly-Guillou and Matthieu Eveillard(Eveillard et al., Journal of Infection 60 (2), S. 154-161, 2010.).Briefly, the mice were rendered transiently neutropenic by injectingcyclophosphamide by intra-peritoneal injection (150 mg/kg body weight in0.15 ml) on days 4 and 3 before A. baumannii inoculation. On the day 0,3 h before A. baumannii inoculation, mice were passively vaccinatedintraperitoneally with either 0.15 ml rabbit antiserum, naïve rabbitserum or PBS. Pneumonia was induced analogous to the active immunizationprotocol starting with anesthetization of the mice. Analogous, survival,clinical score and body weight were monitored. Results are shown inFIGS. 11 and 13.

Example 13: Generation of mAbs

Peripheral blood lymphocytes purified by Ficoll-Paque gradientcentrifugation from 40 ml whole blood samples are resuspended in 3 mlcell culture medium (IMDM/Ham's F12 50:50; 10% FCS) and 3 ml cellculturesupernatant of EBV-secreting B-95-8 marmoset cells. After incubation for3 to 15 hours at 37° C. and 6.5% CO₂, loose and adherent cells aretransferred after one washing/centrifugation step in HANKS buffer into18 ml cell culture medium containing 1 μg/ml Cyclosporin A+/−supplements. Cells are seeded in 96 well round bottom plates in volumesof 200 μl per well and cultivated for 1 to 3 weeks until fast growingcolonies, lymphoblastoid cell lines (LCL), can be identified and themedium turns yellow due to pH shifting. Cell supernatants are analyzedfor antigen-specific antibodies by ELISA. Antibody-producing cells areafterwards passaged until cell numbers sufficient for the followingfusion procedure are obtained. 2.5×10⁵ or 1.25×10⁵ LCL and the sameamount of fusion partner cells (e.g. mouse-human heteromyeloma LA55) areused for one electrofusion. Cells are harvested when growingexponentially and washed once with PBS and afterwards with electrofusionbuffer. The LCL supernatant is stored at 4° C. and later used as apositive control in screening ELISAs. After combining the two celltypes, cells are spinned down and the emerged pellet is carefullyresuspended in 200 μl electrofusion buffer. For fusion, the cell mixtureis transferred to the Helix-Fusion chamber of a Multiporator (Eppendorf)and the cell fusion program (Alignment: 5 Volt, 30 sec; Pulse: 30 Volt,30 sec, No. Pulse: 3; Post-Alignment: 5 Volt, 30 sec) is applied.Afterwards the cells are incubated at room temperature for 5 to 10minutes, resuspended in 4 ml cell culture medium without FCS anddispensed in 4 wells of a 24-well plate. After 3 hours of incubation at37° C. and 6.5% CO₂ the cell suspensions are pooled, mixed with 4 mlselection medium and transferred to a 96-well round-bottom plate (200μl/well). After one week the medium is replaced by cell culture mediumwithout selective reagents. Afterwards cells are cultivated until fastgrowing hybridoma colonies can be identified. Then the supernatants areanalyzed for the presence of specific antibodies by ELISA. Theidentified hybridoma are grown up, re-cloned by two time single cellcultivation and cryopreserved for development.

Example 14: Bactericidal Assay

HL-60 cells (ATCC CCL-240) were cultivated in IMDM (Sigma-Aldrich) orRPMI-1640 (Sigma-Aldrich), each containing 20% (v/v) heat inactivated(40 min at 56° C.) fetal bovine serum (FCS) (Biochrome, Berlin, Germany)and 2 mM GlutaMAX-I (Gibco/Invitrogen, USA) at 37° C. in a 6% CO₂ cellculture incubator. Cells were maintained at a cell density between10⁵-10⁶ cells/ml by passaging cells every 3-4 days into a fresh cellculture flask and replacing 80%-90% of the cell culture with freshmedia. HL-60 cells were not cultivated longer than 4 months.

Four days in advance of the bactericidal assay, the HL-60 cells weredifferentiated by addition of 310 μl dimethylformamide (Sigma-Aldrich,Germany) to 8×10⁶ HL-60 cells in 40 ml medium. The cells were incubatedfor 4 days at 37° C.

On the day of the bactericidal assay, overnight cultures of A. baumanniiin LB were diluted 1:150 in 3 ml fresh LB medium and incubated for 3 hat 37° C. and 200 rpm until an OD₆₀₀ of 0.5-1.5 was reached. The culturewas diluted to an OD₆₀₀ of 3.8×10⁻⁶ in to room temperature prewarmedIMDM containing 0.1 (w/v) % BSA. Antibodies or serum and correspondingcontrols were equally diluted in PBS. Each diluted antibody (20 μl) wascombined with 80 μl bacterial suspension in a well of a 96-well cellculture plate. The concentration of antibody depended on the A.baumannii strain, serum and antibody used. Antibody (0.5 μg/well forATCC 19606 and CsuE KO, 5 μg/well for Ruh134) of total IgG from rabbitimmune serum (□CsuAB) or naive rabbit serum was used.

Antibody and bacteria were incubated at 37° C. and 130 rpm for 20 min.Differentiated HL-60 cells (60 μl) or medium and 20 μl baby rabbit serum(BRS) (Charles River Wiga GMBH, Germany) as complement or BRS previouslyheat inactivated by incubating for 40 min at 56° C. (HBRS) were addedand wells incubated at 37° C. and 130 rpm for 120 min. Colony formingunits (cfu) were determined as follows. Each well was resuspendedthoroughly and 10 μl of undiluted suspension and a 1:5 dilutedsuspension were plated onto LBA. LBA-plates were incubated at 37° C. andcfus were counted 16-20 h later.

Results are shown in FIGS. 9 A and B.

Example 15: Peptide/Epitope Mapping

Peptide mapping of rabbit immune sera and the corresponding pre-immunesera were performed by Pepperprint GmbH (Heidelberg, Germany) bymicroarray analysis. From the Seq ID NOs 2, 4, 6, 8, 10, 12, 14 and 16,all possible linear peptide fragments consisting of 5, 8 and 15 aminoacids were synthesized. Fragments were coated onto PEGMA copolymer filmwith a linker of two β-alanines and aspartic acid. The microarraysconsisting of peptide fragments from Seq ID NOs 2, 4, 6, 8, 10, 12, 14and 16 in duplicate were stained with rabbit pre-immune and specificimmune sera that were raised against the corresponding recombinantproteins (e.g. Microarray coated with peptide fragments of Seq ID NO 2was stained with pre-immune and immune serum of a rabbit immunized withrecombinant protein of Seq ID NO 2). The generation of recombinantproteins is described in Example 3 and 4. The generation of the immunesera is described in Example 5. The antibody staining procedure wasperformed as follows: after 30 min pre-swelling in standard buffer (PBS,pH 7.4+0.05% Tween 20) and 30 min in blocking buffer (Rockland blockingbuffer B-070), the peptide microarrays with the coated peptide fragmentswere incubated with rabbit pre-immune sera at a dilution of 1:1000 for16 h at 4° C. and shaking at 500 rpm. After washing in standard buffertwice for 1 min, the microarrays were stained with the secondary goatanti-rabbit IgG(H+L) DyLight680 antibody at a dilution of 1:5000 for 30min at room temperature. The peptide microarrays were washed twice for 1min with standard buffer, rinsed with distilled water and dried in astream of air. Read-out was done with Odyssey Imaging System at aresolution of 21 μm and green/red intensities of 7/7. After the readout, the staining procedure was repeated with the corresponding immuneserum starting with the pre-swelling step. The incubation in blockingbuffer was skipped. The signal intensities of the correspondingpre-immune and immune sera were compared. A software algorithm from thePepSlide® Analyzer was used to calculate the median staining intensityof each peptide, duplicates averaged and the standard deviationcalculated. Based on average intensities, an intensity map was generatedand specific binders in the peptide map identified. Peptide andintensity maps were correlated with visual inspection of the microarrayscans to identify consensus motifs and distinctive peptides thatinteracted specifically with the rabbit immune sera.

Results Example 15

To verify the immunogenicity of peptide fragments, microarray analysiswas performed as described in Example 15. The Seq ID NOs 2, 4, 6, 8, 10,12, 14 and 16 were translated into linear peptide fragments consistingof 5, 8 and 15 amino acids and interaction analyzed with specific rabbitimmune sera. By this approach for all rabbit immune sera, antibodyepitopes were identified with varying lengths. Most consensus motifsconsisted of 5 amino acids, while others were 6, 7 or 8 amino acids inlength. The pre-immune sera used as control showed only negligiblebackground. Based on the fragment, consisting of 8 amino acids, theimmune serum specific to Seq ID NO 14 showed a single epitope consensusmotif PVDFTVAI (SEQ ID NO: 36) and thus shows monoclonal reactivity.

1-35. (canceled)
 36. An antibody generated by vaccination with apolypeptide comprising the sequence of SEQ ID NO:
 8. 37. The antibody ofclaim 36, wherein said antibody induces an effector function towardsAcinetobacter baumanii.
 38. The antibody of claim 37, wherein theantibody is polyclonal.
 39. The antibody of claim 37, wherein theantibody is monoclonal.
 40. The antibody of claim 39, wherein theantibody specifically binds the epitope motif FEDFN.
 41. The antibody ofclaim 40, wherein the antibody is humanized.
 42. The antibody of claim36, in a eukaryotic expression host cell adapted to express theantibody.
 43. The antibody of claim 36, in a hybridoma host cellexpressing the antibody.
 44. The antibody of claim 36, wherein thevaccination is enhanced by an adjuvant.
 45. An antibody produced byintroducing a polypeptide comprising the sequence of SEQ ID NO: 8 into amammal along with an adjuvant; wherein the antibody binds to the motifFEDFN and induces an effector function towards Acinetobacter baumanii.